Optical biosensors

ABSTRACT

Provided are biosensors, compositions comprising biosensors, and methods of using biosensors in living cells and organisms. The biosensors are able to be selectively targeted to certain regions or structures within a cell. The biosensors may provide a signal when the biosensor is targeted and/or in response to a property of the cell or organism such as membrane potential, ion concentration or enzyme activity.

PRIORITY

This application is a U.S. National Stage Application based onInternational Application Serial No. PCT/US2008/051962 filed 24 Jan.2008 and claims the benefit of U.S. Provisional Application Ser. No.60/897,120 filed Jan. 24, 2007 and U.S. Provisional Application Ser. No.61/013,098 filed Dec. 12, 2007, the contents of each of which areincorporated by reference in their entirety.

GOVERNMENT SUPPORT

The subject invention was made in part with support from the U.S.Government under Grant Number 1-U54-RR022241 awarded by the NIH.Accordingly, the U.S. Government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING

This Application contains a Sequence Listing in accordance with 37C.F.R. §§1.821-1.825. The material in the Sequence Listing text file isherein incorporated by reference in its entirety in accordance with 37C.F.R. §1.52(e)(5). The Sequence Listing, entitled “070683PCTUS Jul. 24,2012 ST25.txt”, contains one 111 Kb text file and was created on Jul.22, 2009 and amended on Jul. 24, 2012 using an IBM-PC machine format.

BACKGROUND

The identification, analysis and-monitoring of biological analytes (suchas polypeptides, polynucleotides, polysaccharides and the like) orenvironmental analytes (such as pesticides, biowarfare agents, foodcontaminants and the like) has become increasingly important forresearch and industrial applications. Conventionally, analyte detectionsystems are based on analyte-specific binding between an analyte and ananalyte-binding receptor. Such systems typically require complexmulticomponent detection systems (such as ELISA sandwich assays) orelectrochemical detection systems, or require that both the analyte andthe receptor are labeled with detection molecules (for examplefluorescence resonance energy transfer or FRET systems).

One method for detecting analyte-binding agent interactions involves asolid phase format employing a reporter labeled analyte-binding agentwhose binding to or release from a solid surface is dependent on thepresence of analyte. In a typical solid-phase sandwich type assay, forexample, the analyte to be measured is an analyte with two or morebinding sites, allowing analyte binding both to a receptor carried on asolid surface, and to a reporter-labeled second receptor. The presenceof analyte is detected based on the presence of the reporter bound tothe solid surface.

A variety of devices for detecting analyte/receptor interactions arealso known. The most basic of these are purely chemical/enzymatic assaysin which the presence or amount of analyte is detected by measuring orquantitating a detectable reaction product, such as goldimmunoparticles. Analyte/receptor interactions can also be detected andquantitated by radiolabel assays. Quantitative binding assays of thistype involve two separate components: a reaction substrate, e.g., asolid-phase test strip and a separate reader or detector device, such asa scintillation counter or spectrophotometer. The substrate is generallyunsuited to multiple assays, or to miniaturization, for handlingmultiple analyte assays from a small amount of body fluid sample.

Biosensor devices integrate the assay substrate and detector surfaceinto a single device. One general type of biosensor employs an electrodesurface in combination with current or impedance measuring elements fordetecting a change in current or impedance in response to the presenceof a ligand-receptor binding event. This type of biosensor is disclosed,for example, in U.S. Pat. No. 5,567,301. Gravimetric biosensors employ apiezoelectric crystal to generate a surface acoustic wave whosefrequency, wavelength and/or resonance state are sensitive to surfacemass on the crystal surface. The shift in acoustic wave properties istherefore indicative of a change in surface mass, e.g.; due to aligand-receptor binding event. U.S. Pat. Nos. 5,478,756 and 4,789,804describe gravimetric biosensors of this type. Biosensors based onsurface plasmon resonance (SPR) effects have also been proposed, forexample, in U.S. Pat. Nos. 5,485,277 and 5,492,840. These devicesexploit the shift in SPR surface reflection angle that occurs withperturbations, e.g., binding events, at the SPR interface. Finally, avariety of biosensors that utilize changes in optical properties at abiosensor surface are known, e.g., U.S. Pat. No. 5,268,305.

All of the above analyte detection systems are characterized by therequirement for a secondary detection system to monitor interactionsbetween the analyte and the receptor. A need still exists for a direct,homogeneous assay for analyte detection, i.e., one that may be used inliving cells, which will be more versatile in terms of the range ofapplications and devices with which it can be used.

SUMMARY

Provided are biosensors, compositions comprising biosensors, and methodsof using biosensors in living cells and organisms. The biosensors areable to be selectively targeted to certain regions or structures withina cell. The biosensors may provide a signal when the biosensor istargeted and/or in response to a property of the cell or organism suchas membrane potential, ion concentration or enzyme activity.

In one embodiment, the biosensors comprise at least two components; (1)a selectivity component capable of interacting with a target molecule ofinterest and a (2) reporter molecule that produces a detectable changein signal upon interaction of the selectivity component with the targetmolecule. The reporter molecule may be covalently linked to theselectivity component, or it may be able to noncovalently interact withthe selectivity component. In certain embodiments, the selectivitycomponent is a ligand of a reporter molecule such as a dye.

The selectivity component, which in certain embodiments is expressedwithin the cell or organism to be analyzed, may be a polypeptide(including antibodies and non-antibody receptor molecules, and fragmentsand variants thereof), polynucleotide (including aptamers), templateimprinted material, or organic and inorganic binding element. Theselectivity component may be biologically selected to favor reportermolecule binding and sensitivity. The selectivity component may binddirectly to a target molecule, or be fused to a targeting moiety (suchas a protein) that binds to the target molecule.

The reporter molecule may be sensitive to changes in the environment,including, for example, pH sensitive molecules, polarity sensitivemolecules, restriction sensitive molecules, or mobility sensitivemolecules. The reporter molecule may, in embodiments where in itnoncovalently interacts with the selectivity component, comprise anadditional moiety that binds to the selectivity component.

The biosensor may optionally comprise a chemical handle suitable tofacilitate isolation, immobilization, identification, or detection ofthe biosensors and/or which increases the solubility of the biosensors.

In one example, the selectivity component is a single chain antibody(scFv) that comprises amino acid sequences that lead to specific bindingof certain reporter molecules such as monomethin cyanine dyes (TO1 andits analogs). In yet other embodiments, the selectivity component is asingle chain antibody that comprises amino acid sequences that lead tospecific binding of a reporter molecule such as Malachite Green (and itsanalogs). The formation of such protein-dye complexes produces a largeincrease in fluorescence of the dye (i.e., fluorogen reporter molecule)when it is in the bound state, thereby allowing detection of binding. Inother examples, the single chain antibody is coupled (e.g., as a fusionprotein or chemical conjugate) to a molecule of interest, such as forexample a cell cycle regulatory protein.

In certain other embodiments, a binary biosensor is used to detect amolecule of interest. For example, a V_(H) chain of a dye-specificantibody can be conjugated to a lipid, sugar, protein or polypeptide ofinterest, while the corresponding V_(L) chain can be coupled to anotherpotential ligand or polypeptide of interest. When the target and ligandare in close proximity, the V_(H) and V_(L) chains become close enoughto form a binding epitope for the dye, a detectable signal is produced.

The biosensors described herein are useful for both in vivo and in vitroapplications. In various embodiments, the biosensors may be used fordetecting one or more target molecules, detecting environmentalpollutants, detecting chemical or biological warfare agents, detectingfood contaminants, and detecting hazardous substances. In an exemplaryembodiment, the biosensors may be used for intracellular monitoring ofone or more target molecules. In such embodiments, at least onecomponent of the biosensor may be expressed within the cell to beanalyzed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the DNA sequence of the construct encoding scFv1 (SEQ IDNO:1). FIG. 1B depicts the protein sequence of the construct encodingscFv1 (SEQ ID NO:2). FIG. 1C is a schematic of the construct encodingscFv1. FIG. 1D depicts the DNA sequence (SEQ ID NO:1) of FIG. 1A withregions of the construct encoding scFv1 of FIG. 1C mapped onto the DNAsequence (SEQ ID NO:1).

FIG. 2 shows various fluorescent dye structures.

FIG. 3 depicts a reporter-molecule localizing system based on sAbtechnology.

FIG. 4 depicts a photoreactive hapten with PEG linker and reportermolecule X.

FIG. 5 depicts a photoreversible hapten with PEG linker and reportermolecule X.

FIG. 6 depicts the structure of Malachite Green derivatized with a PEGamine. In some embodiments, the amino group may be covalently modifiedwith a biotin group for streptavidin coated magnetic bead enrichment ofyeast bearing scFv proteins that bind to the Malachite Green on theopposite end of the PEG linker.

FIG. 7 depicts the structure of Thiazole Orange 1 (TO1) derivatized witha PEG amine. In some embodiments, the amino group may be covalentlymodified with a biotin group for streptavidin coated magnetic beadenrichment of yeast bearing scFv proteins that bind to the TO1 on theopposite end of the PEG linker.

FIG. 8 contains a table listing the peptide sequences of the scFvscomprising the FBPs of Example 5 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15,17, 19 and 21, corresponding to tableentries >HL1-TO1, >HL2-TO1, >HL1.0.1-TO1, >HL1.1-TO1, >HL4-MG, >L5-MG, >H6-MG, >HL7-MG, >H8-MG,and >HL9-MG, respectively).

FIG. 9 contains a table describing the composition and properties of theFBPs of Example 5.

FIGS. 10A and 10B are graphs depicting the fluorescence characterizationof purified FBPs. FIG. 10A depicts the fluorescence spectra ofFBP/fluorogen complexes. Excitation and emission spectra are determinedin the presence of excess purified FBP (2 μM HL 1.0.1-TO 1 and 100 nM TO1-2p; 2 μM HL4-MG or L5-MG and 200 nM MG-2p). The relative fluorescenceof FBP/fluorogen complexes and free fluorogen is determined at fixedexcitation and emission on a microplate reader (the 488 nm laserexcitation is shown as a dotted line). FIG. 10B depicts fluorescencespectra of FBP/fluorogen complexes using different fluorogen analogswhere the depicted R-groups are substituted as the MG-2p R-group. Theexcitation spectra are determined using 2 μM purified HL4MG or L5-MG and200 nM of indicated fluorogen analog: (I) malachite green diethylester;(II) crystal violet; (III) malachite green. Fluorescence intensity isnormalized; actual fluorescence signal varies, mainly due to differencesin binding affinity. Depicted FBP/fluorogen complexes showed 70 to12,000-fold fluorescence enhancement over free fluorogen.

FIGS. 11A, 11B and 11C illustrate fluorogen embodiments and their usewith yeast displayed scFvs. FIG. 11A depicts the structure of thefluorogenic dyes thiazole orange derivative (TO1-2p) and malachite greenderivative (MG-2p) used in various embodiments. FIG. 11B is a graphdepicting the isolation of FBPs using FACS. The Sorting screen showsseparation of yeast cells bearing malachite green—activating scFvs froma bulk yeast population. The horizontal axis shows distribution of cellsby green fluorescence of antibody reagent that labels the c-myc epitope;and the vertical axis depicts distribution of cells by red fluorescencegenerated by binding of MG fluorogen. Sorting window (I) collects cellsenriched for FBPs composed of heavy chain (VH), light chain (VL) andc-myc epitope (M). Sorting window (II) collects cells enriched for FBPscomposed only of the heavy chain. FIG. 11C is a graph depicting ahomogenous format assay of live yeast cells displaying FBPs. Thefluorescence excitation spectrum of displayed HL4-MG is taken on a96-well microplate reader (10⁷ yeast cells in 200 ml effectiveconcentration B10 nM scFv are treated with 200 nM MG-2p). The insetillustrates low levels of fluorescence background signal with JAR200control cells that do not express FBPs.

FIG. 12 depicts the improvement of binding affinity and intrinsicbrightness of HL1-TO1 by directed evolution. Affinity and total cellularbrightness are measured using yeast cell surface displayed scFvs. Totalcellular brightness is measured at saturating fluorogen concentration ona Tecan Safire 2 plate reader, and intrinsic brightness calculated bynormalizing total signal to the relative number of scFvs, determinedseparately by FACS analysis of immunolabeled c-myc epitope. The bargraph depicts relative intrinsic brightness for selected scFvs employedafter one or two generations of directed evolution. Numbers on the barsrepresent cell surface binding K_(D) (nM). The sequence alignments showthe distribution of acquired mutations within the heavy chain variableregion of HL1-TO1 (SEQ ID NO: 73, a fragment corresponding to residues1-121 of SEQ ID NO: 3). Complementarity Determining Regions (“CDRs”)within HL1-TO1 implicated in antigen recognition are underlined andnumbered 1, 2 and 3, (corresponding to SEQ ID NOs 76, 77 and 78,respectively) as identified in the IMGT/V-QUEST database. Amino acidreplacements in bold depict residues found in multiple instances withineach family of improved descendants (a fragment of HL1.1-TO1, SEQ ID NO:74 and a fragment of HL1.0.1-TO1, SEQ ID NO: 75). The dominantreplacements tend to accumulate in CDRs, wherein replacement CDRs withinthe fragment of HL1.1-TO1 shown to align with SEQ. ID NOs: 77 and 78 arerepresented by SEQ ID NOs: 79 and 80 and replacement CDRs within thefragment of HL1.0.1-TO1 shown to align with SEQ. ID NOs: 77 and 78 arerepresented by SEQ ID NOs: 81 and 82. For HL1-TO1, accumulation ofdominant replacements occurs in the heavy chain rather than the lightchain. Among 16 unique second generation descendants that are analyzed,8 positions in the heavy chain accumulate dominant mutations but only 1position in the light chain accumulates dominant mutations. For theselected clones, it can be seen that the first generation replacementsimprove both affinity and brightness, whereas second generationreplacements improve only affinity.

FIG. 13 is an SDS-PAGE gel of purified FBPs with 1 μg of BCA quantitatedscFv loaded per lane, where lane-1 is loaded with a MW standard; lane-2is loaded with HL1.0.1-TO1; lane-3 is loaded with HL4-MG; lane-4 isloaded with L %-MG; and lane-5 is loaded with H6-MG.

FIG. 14 provides graphs illustrating fluorogen binding to yeastdisplayed FBPs (above dashed line) and soluble FBPs (below dashed line).Provided are representative binding curves with 95% confidence intervalsfor each. One-site hyperbolic saturation binding analysis was applied toall displayed FBPs (above dashed line) and soluble H6-MG and asaturation binding with ligand depletion algorithm was applied to othersoluble FBPs (below dashed line).

FIG. 15 provides graphs illustrating absorbance of fluorogens andFBP/fluorogen complexes. Shown are samples used in quantum yielddeterminations. The absorbance of FBP/fluorogen complexes is obtained ona dual beam PerkinElmer Lambda 45 spectrophotometer using an equalconcentration of FBP without fluorogen as the reference.

FIGS. 16A, 16B and 16C are graphs depicting photobleaching curves forFBPs. FIG. 16A is a graph of photobleaching curves for TO1-FBP and EGFPdisplayed on yeast. JAR200 yeast strains displaying HL1.0.1-TO1 and EGFPare immobilized on concanavalin-A treated 35 mm petri dishes with 14 mmoptical microwell (MatTek Corp) and bleached in 2 ml modified PBS usingan Olympus IX50 inverted microscope equipped with a 100 W Hg lamp,40×1.3 NA oil objective and a Photometrics CoolSnap HQ camera withHQ470/40 excitation and HQ500 LP emission filters (Chroma set #41018,total irradiance at the specimen plane was measured at 30 mW (13.6μW/μm2)). Each curve represents an average of scans of 8-12 individualcells. Fluorescence of EGFP is normalized (scaled down ˜2.5-fold) tomatch HL1.0.1-TO1 cells visualized with 375 nM TO1-2p. FIG. 16B is agraph depicting the photobleaching lifetime of yeast displayed TO1-FBPand EGFP. JAR200 yeast cells treated are bleached on a Leica DMI 6000 Bconfocal microscope using 488 nm laser excitation at 100% power andmonitoring emission with a 500-600 nm window. Data from individual cellsare averaged and the EGFP signal is normalized (scaled down ˜3-fold) tomatch initial HL1.0.1-TO1 fluorescence. Plotted data points aredisplayed with a single exponential decay curve (Graphpad Prism 4.0software). Lifetimes are corrected by comparing excitation intensitiesof these cells at 488 nm to the intensity at their excitation maxima(EGFP at 502 nm, HL1.0.1-TO1 at 512 nm), determined on a Tecan Safire2plate reader. FIG. 16C is a graph depicting the photobleaching of MG-FBPdisplayed on mammalian cells. NIH 3T3 cells stably expressing bothHL4-MG and HL1.1-TO1 simultaneously are isolated using FACS, and grownas a layer on the optical window of 35 mm petri dishes. Bleachingexperiments are carried out in PBS w/Ca and Mg using HQ620/60 excitationand HQ665 LP emission filters (Chroma set #41024), total specimen planeirradiance is measured at 30 mW.

FIG. 17 is a graph depicting the effect of fluorogens on yeast cellgrowth. JAR200 cells are inoculated at ˜10⁶ cells/ml into 35 ml SD+CAAmedium in 125 ml baffled flasks and allowed to grow at 30° C. at 300 RPMfor 2 hours prior to addition of fluorogens at the indicatedconcentrations. One ml samples are removed at indicated time points, andgrowth halted by addition of 75 μl of 300 mM NaN₃ prior to readingabsorbance. Doubling time of about 1.9 hrs is unchanged by mostfluorogen treatments. For 500 nM MG-ester, the doubling time is about2.8 hrs; and for 500 nM MG, the doubling time is over 24 hrs.

FIG. 18A is a binding schematic for the FBP L5-MG. FIG. 18B is a bindingschematic for the FBP H8-MG.

FIG. 19A is a binding schematic for the FBP H6-MG. FIG. 19B is a bindingschematic for the FBP HL4-MG.

FIG. 20A is a binding schematic for the FBP HL1-TO1. FIG. 20B is abinding schematic for the FBP HL1.0.1-TO1.

FIG. 21A is a binding schematic for the FBP HL1.1-TO1. FIG. 21B is abinding schematic for the FBP HL7-MG.

FIG. 22A is a binding schematic for the FBP HL2-TO1. FIG. 22B is abinding schematic for the FBP HL9-MG.

DETAILED DESCRIPTION

To provide an overall understanding, certain illustrative embodimentswill now be described; however, it will be understood by one of ordinaryskill in the art that the systems and methods described herein can beadapted and modified to provide systems and methods for other suitableapplications and that other additions and modifications can be madewithout departing from the scope of the systems and methods describedherein.

Unless otherwise specified, the illustrated embodiments can beunderstood as providing exemplary features of varying detail of certainembodiments, and therefore unless otherwise specified, features,components, modules, and/or aspects of the illustrations can becombined, separated, interchanged, and/or rearranged without departingfrom the disclosed systems or methods.

1. Introduction

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art.

Other than in the examples herein, or unless otherwise expresslyspecified, all of the numerical ranges, amounts, values and percentages,such as those for amounts of materials, elemental contents, times andtemperatures of reaction, ratios of amounts, and others, in thefollowing portion of the specification and attached claims, may be readas if prefaced by the word “about” even though the term “about” may notexpressly appear with the value, amount, or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains errornecessarily resulting from the standard deviation found in itsunderlying respective testing measurements. Furthermore, when numericalranges are set forth herein, these ranges are inclusive of the recitedrange end points (i.e., end points may be used). When percentages byweight are used herein, the numerical values reported are relative tothe total weight.

Also, it should be understood that any numerical range recited herein isintended to include all sub-ranges subsumed therein. For example, arange of “1 to 10” is intended to include all sub-ranges between (andincluding) the recited minimum value of 1 and the recited maximum valueof 10, that is, having a minimum value equal to or greater than 1 and amaximum value of equal to or less than 10. The articles “a” and “an” areused herein to refer to one or to more than one (i.e., to at least one)of the grammatical object of the article. By way of example, “anelement” means one element or more than one element.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and allstereoisomers of any of any of the foregoing.

As used herein, the term “selectivity component” refers to a moleculecapable of interacting with a target molecule. Selectivity componentshaving limited cross-reactivity are generally preferred. In certainembodiments, suitable selectivity components include, for example,polypeptides, such as for example, antibodies, monoclonal antibodies, orderivatives or analogs thereof, including without limitation: Fvfragments, single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2fragments, single domain antibodies, camelized antibodies and antibodyfragments, humanized antibodies and antibody fragments, and multivalentversions of the foregoing; multivalent binding reagents includingwithout limitation: monospecific or bispecific antibodies, such asdisulfide stabilized Fv fragments, scFv tandems ((scFv)₂ fragments),diabodies, tribodies or tetrabodies, which typically are covalentlylinked or otherwise stabilized (i.e., leucine zipper or helixstabilized) scFv fragments; and other binding reagents including, forexample, aptamers, template imprinted materials (such as those of U.S.Pat. No. 6,131,580), and organic or inorganic binding elements. Inexemplary embodiments, a selectivity component specifically interactswith a single epitope. In other embodiments, a selectivity component mayinteract with several structurally related epitopes.

The term “ligand” refers to a binding moiety for a specific targetmolecule. The molecule can be a cognate receptor, a protein, a smallmolecule, a hapten, or any other relevant molecule.

The term “antibody” refers to an immunoglobulin, derivatives thereofwhich maintain specific binding ability, and proteins having a bindingdomain which is homologous or largely homologous to an immunoglobulinbinding domain. As such, the antibody operates as a ligand for itscognate antigen, which can be virtually any molecule. Natural antibodiescomprise two heavy chains and two light chains and are bi-valent. Theinteraction between the variable regions of heavy and light chain formsa binding site capable of specifically binding an antigen. The term“V_(H)” refers to a heavy chain variable region of an antibody. The term“V_(L)” refers to a light chain variable region of an antibody.Antibodies may be derived from natural sources, or partly or whollysynthetically produced. An antibody may be monoclonal or polyclonal. Theantibody may be a member of any immunoglobulin class, including any ofthe human classes: IgG, IgM, IgA, IgD, and IgE. In exemplaryembodiments, antibodies used with the methods and compositions describedherein are derivatives of the IgG class.

The term “antibody fragment” refers to any derivative of an antibodywhich is less than full-length. In exemplary embodiments, the antibodyfragment retains at least a significant portion of the full-lengthantibody's specific binding ability. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, Fv, dsFv, scFv,diabody, and Fd fragments. The antibody fragment may be produced by anymeans. For instance, the antibody fragment may be enzymatically orchemically produced by fragmentation of an intact antibody, it may berecombinantly or partially synthetically produced. The antibody fragmentmay optionally be a single chain antibody fragment. Alternatively, thefragment may comprise multiple chains which are linked together, forinstance, by disulfide linkages. The fragment may also optionally be amultimolecular complex. A functional antibody fragment will typicallycomprise at least about 50 amino acids and more typically will compriseat least about 200 amino acids.

The term “Fab” refers to an antibody fragment that is essentiallyequivalent to that obtained by digestion of immunoglobulin (typicallyIgG) with the enzyme papain. The heavy chain segment of the Fab fragmentis the Fd piece. Such fragments may be enzymatically or chemicallyproduced by fragmentation of an intact antibody, recombinantly producedfrom a gene encoding the partial antibody sequence, or it may be whollyor partially synthetically produced. Methods for preparing Fab fragmentsare known in the art. See, for example, Tijssen, Practice and Theory ofEnzyme Immunoassays (Elsevier, Amsterdam, 1985).

The term “Fab′” refers to an antibody fragment that is essentiallyequivalent to that obtained by reduction of the disulfide bridge orbridges joining the two heavy chain pieces in the F(ab′)₂ fragment. Suchfragments may be enzymatically or chemically produced by fragmentationof an intact antibody, recombinantly produced from a gene encoding thepartial antibody sequence, or it may be wholly or partiallysynthetically produced.

The term “F(ab′)₂” refers to an antibody fragment that is essentiallyequivalent to a fragment obtained by digestion of an immunoglobulin(typically IgG) with the enzyme pepsin at pH 4.0-4.5. Such fragments maybe enzymatically or chemically produced by fragmentation of an intactantibody, recombinantly produced from a gene encoding the partialantibody sequence, or it may be wholly or partially syntheticallyproduced.

The term “Fv” refers to an antibody fragment that consists of one V_(H)and one V_(L) domain held together by noncovalent interactions. The term“dsFv” is used herein to refer to an Fv with an engineeredintermolecular disulfide bond to stabilize the V_(H)-V_(L) pair. Methodsfor preparing Fv fragments are known in the art. See, for example, Mooreet al., U.S. Pat. No. 4,462,334; Hochman et al., Biochemistry 12: 1130(1973); Sharon et al., Biochemistry 15: 1591 (1976); and Ehrlich et al.,U.S. Pat. No. 4,355,023.

The terms “single-chain Fvs” and “scFvs” refers to recombinant antibodyfragments consisting of only the variable light chain (V_(L)) andvariable heavy chain (V_(H)) covalently connected to one another by apolypeptide linker. Either V_(L) or V_(H) may be the NH₂-terminaldomain. The polypeptide linker may be of variable length and compositionso long as the two variable domains are bridged without serious stericinterference. In exemplary embodiments, the linkers are comprisedprimarily of stretches of glycine and serine residues with some glutamicacid or lysine residues interspersed for solubility. Methods forpreparing scFvs are known in the art. See, for example, PCT/US/87/02208and U.S. Pat. No. 4,704,692.

The term “single domain antibody” or “Fd” refers to an antibody fragmentcomprising a V_(H) domain that interacts with a given antigen. An Fddoes not contain a V_(L) domain, but may contain other antigen bindingdomains known to exist in antibodies, for example, the kappa and lambdadomains. In certain embodiments, the Fd comprises only the F_(L)component. Methods for preparing Fds are known in the art. See, forexample, Ward et al., Nature 341:644-646 (1989) and EP 0368684 A1.

The term “single chain antibody” refers to an antibody fragment thatcomprises variable regions of the light and heavy chains joined by aflexible linker moiety. Methods for preparing single chain antibodiesare known in the art. See, for example, U.S. Pat. No. 4,946,778 toLadner et al.

The term “diabodies” refers to dimeric scFvs. The components ofdiabodies typically have shorter peptide linkers than most scFvs andthey show a preference for associating as dimers. The term diabody isintended to encompass both bivalent (i.e., a dimer of two scFvs havingthe same specificity) and bispecific (i.e., a dimer of two scFvs havingdifferent specificities) molecules. Methods for preparing diabodies areknown in the art. See, for example, EP 404097 and WO93/11161.

The term “triabody” refers to trivalent constructs comprising 3 scFv's,and thus comprising 3 variable domains (see, e.g., Iliades et al., FEBSLett. 409(3):43741 (1997)). Triabodies is meant to include moleculesthat comprise 3 variable domains having the same specificity, or 3variable domains wherein two or more of the variable domains havedifferent specificities.

The term “tetrabody” refers to engineered antibody constructs comprising4 variable domains (see, e.g., Pack et al., J Mol Biol. 246(1): 28-34(1995) and Coloma & Morrison, Nat Biotechnol. 15(2): 159-63 (1997)).Tetrabodies is meant to include molecules that comprise 4 variabledomains having the same specificity, or 4 variable domains wherein twoor more of the variable domains have different specificities.

The term “camelized antibody” refers to an antibody or variant thereofthat has been modified to increase its solubility and/or reduceaggregation or precipitation. For example; camelids produce heavy-chainantibodies consisting only of a pair of heavy chains wherein the antigenbinding site comprises the N-terminal variable region or V_(HH)(variable domain of a heavy chain antibody). The V_(HH) domain comprisesan increased number of hydrophilic amino acid residues that enhance thesolubility of a V_(HH) domain as compared to a V_(H) region fromnon-camelid antibodies. Camelization of an antibody or variant thereofinvolves replacing one or more amino acid residues of a non-camelidantibody with corresponding amino residues from a camelid antibody.

As used herein, the term “epitope” refers to a physical structure on amolecule that interacts with a selectivity component, such as anantibody. In exemplary embodiments, epitope refers to a desired regionon a target molecule that specifically interacts with a selectivitycomponent.

“Interact” is meant to include detectable interactions betweenmolecules, such as may be detected using, for example, a hybridizationassay. Interact also includes “binding” interactions between molecules.Interactions may be, for example, protein-protein, protein-nucleic acid,protein-small molecule or small molecule-nucleic acid, and includes forexample, antibody-antigen binding, receptor-ligand binding,hybridization, and other forms of binding. In certain embodiments, aninteraction between a ligand and a specific target will lead to theformation of a complex, wherein the ligand and the target are unlikelyto dissociate. Such affinity for a ligand and its target can be definedby the dissociation constant (K_(d)) as known in the art. A complex mayinclude a ligand for a specific dye and is referred to herein as a“ligand-dye” complex.

The term “immunogen” traditionally refers to compounds that are used toelicit an immune response in an animal, and is used as such herein.However, many techniques used to produce a desired selectivitycomponent, such as the phage display and aptamer methods describedbelow, do not rely wholly, or even in part, on animal immunizations.Nevertheless, these methods use compounds containing an “epitope,” asdefined above, to select for and clonally expand a population ofselectivity components specific to the “epitope.” These in vitro methodsmimic the selection and clonal expansion of immune cells in vivo, and,therefore, the compounds containing the “epitope” that is used toclonally expand a desired population of phage, aptamers and the like invitro are embraced within the definition of “immunogens.”

Similarly, the terms “hapten” and “carrier” have specific meaning inrelation to the immunization of animals, that is, a “hapten” is a smallmolecule that contains an epitope, but is incapable as serving as animmunogen alone. Therefore, to elicit an immune response to the hapten,the hapten is conjugated with a larger carrier, such as bovine serumalbumin or keyhole limpet hemocyanin, to produce an immunogen. Apreferred immune response would recognize the epitope on the hapten, butnot on the carrier. As used herein in connection with the immunizationof animals, the terms “hapten” and “carrier” take on their classicaldefinition. However, in the in vitro methods described herein forpreparing the desired binding reagents, traditional “haptens” and“carriers” typically have their counterpart in epitope-containingcompounds affixed to suitable substrates or surfaces, such as beads andtissue culture plates.

The term “aptamer” refers to a nucleic acid molecule that mayselectively interact with a non-oligonucleotide molecule or group ofmolecules. In various embodiments, aptamers may include single-stranded,partially single-stranded, partially double-stranded or double-strandednucleic acid sequences; sequences comprising nucleotides,ribonucleotides, deoxyribonucleotides, nucleotide analogs, modifiednucleotides and nucleotides comprising backbone modifications,branchpoints and normucleotide residues, groups or bridges; syntheticRNA, DNA and chimeric nucleotides, hybrids, duplexes, heteroduplexes;and any ribonucleotide, deoxyribonucleotide or chimeric counterpartthereof and/or corresponding complementary sequence. In certainembodiments, aptamers may include promoter or primer annealing sequencesthat may be used to amplify, transcribe or replicate all or part of theaptamer.

As used herein, the term “reporter molecule” refers to a moleculesuitable for detection, such as, for example, spectroscopic detection.Examples of reporter molecules include, but are not limited to, thefollowing: fluorescent labels, enzymatic labels, biotinyl groups, andpredetermined polypeptide epitopes recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags). Examples and use ofsuch reporter molecules are described in more detail below. In someembodiments, reporter molecules are attached by spacer arms of variouslengths to reduce potential steric hindrance. Reporter molecules may beincorporated into or attached (including covalent and non-covalentattachment) to a molecule, such as a selectivity component. Variousmethods of labeling polypeptides are known in the art and may be used.

As used herein, the term “sensor dye” refers to a reporter molecule thatexhibits an increase, decrease or modification of signal in response toa change in the environment. In exemplary embodiments, the sensor dye isa fluorescent molecule that is responsive to changes in polarity and/ormobility of the dye, as well as, the changes microenvironment pH and/orviscosity, or combinations thereof.

A “fusion protein” or “fusion polypeptide” refers to a chimeric proteinas that term is known in the art and may be constructed using methodsknown in the art. In many examples of fusion proteins, there are twodifferent polypeptide sequences, and in certain cases, there may bemore. The sequences may be linked in frame. A fusion protein may includea domain which is found (albeit in a different protein) in an organismwhich also expresses the first protein, or it may be an “interspecies”,“intergenic”, etc. fusion expressed by different kinds of organisms. Invarious embodiments, the fusion polypeptide may comprise one or moreamino acid sequences linked to a first polypeptide. In the case wheremore than one amino acid sequence is fused to a first polypeptide, thefusion sequences may be multiple copies of the same sequence, oralternatively, may be different amino acid sequences. The fusionpolypeptides may be fused to the N-terminus, the C-terminus, or the N-and C-terminus of the first polypeptide. Exemplary fusion proteinsinclude polypeptides comprising a glutathione S-transferase tag(GST-tag), histidine tag (His-tag), an immunoglobulin domain or animmunoglobulin binding domain.

As used herein, the term “array” refers to a set of selectivitycomponents immobilized onto one or more substrates so that eachselectivity component is at a known location. In an exemplaryembodiment, a set of selectivity components is immobilized onto asurface in a spatially addressable manner so that each individualselectivity component is located at a different and identifiablelocation on the substrate.

The term “chemical handle” refers to a component that may be attached toa biosensor as described herein so as to facilitate its isolation,immobilization, identification, or detection and/or which increases itssolubility. Suitable chemical handles include, for example, apolypeptide, a polynucleotide, a carbohydrate, a polymer, or a chemicalmoiety and combinations or variants thereof.

The term “conserved residue” refers to an amino acid that is a member ofa group of amino acids having certain common properties. The term“conservative amino acid substitution” refers to the substitution(conceptually or otherwise) of an amino acid from one such group with adifferent amino acid from the same group. A functional way to definecommon properties between individual amino acids is to analyze thenormalized frequencies of amino acid changes between correspondingproteins of homologous organisms. According to such analyses, groups ofamino acids may be defined where amino acids within a group exchangepreferentially with each other, and therefore resemble each other mostin their impact on the overall protein structure. One example of a setof amino acid groups defined in this manner include: (i) a chargedgroup, consisting of Glu and Asp, Lys, Arg and His, (ii) apositively-charged group, consisting of Lys, Arg and His, (iii) anegatively-charged group, consisting of Glu and Asp, (iv) an aromaticgroup, consisting of Phe, Tyr and Trp, (v) a nitrogen ring group,consisting of His and Trp, (vi) a large aliphatic nonpolar group,consisting of Val, Leu and Ile, (vii) a slightly-polar group, consistingof Met and Cys, (viii) a small-residue group, consisting of Ser, Thr,Asp, Asn, Gly, Ala, Glu, Gln and Pro, (ix) an aliphatic group consistingof Val, Leu, Ile, Met and Cys, and (x) a small hydroxyl group consistingof Ser and Thr.

“Isolated”, with respect to nucleic acids, such as DNA or RNA, refers tomolecules separated from other DNAs, or RNAs, respectively, that arepresent in the natural source of the macromolecule. Isolated also refersto a nucleic acid or peptide that is substantially free of cellularmaterial, viral material, or culture medium when produced by recombinantDNA techniques, or chemical precursors or other chemicals whenchemically synthesized. Moreover, an “isolated nucleic acid” is meant toinclude nucleic acid fragments which are not naturally occurring asfragments and would not be found in the natural state. “Isolated” alsorefers to polypeptides which are isolated from other cellular proteinsand is meant to encompass both purified and recombinant polypeptides.

The term “mammal” is known in the art, and exemplary mammals includehumans, primates, bovines, porcines, canines, felines, and rodents(e.g., mice and rats).

The term “microenvironment” refers to localized conditions within alarger area. For example, association of two molecules within a solutionmay alter the local conditions surrounding the associating moleculeswithout affecting the overall conditions within the solution.

The term “nucleic acid” refers to a polymeric form of nucleotides,either ribonucleotides or deoxynucleotides or a modified form of eithertype of nucleotide. The terms should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single-stranded(such as sense or antisense) and double-stranded polynucleotides.

The term “polypeptide”, and the terms “protein” and “peptide” which areused interchangeably herein, refers to a polymer of amino acids.

The terms “polypeptide fragment” or “fragment”, when used in regards toa reference polypeptide, refers to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thecorresponding positions in the reference polypeptide. Such deletions mayoccur at the amino-terminus or carboxy-terminus of the referencepolypeptide, or alternatively both. Fragments typically are at least 5,10, 20, 50, 100, 500 or more amino acids long. A fragment can retain oneor more of the biological activities of the reference polypeptide.

The term “sequence homology” refers to the proportion of base matchesbetween two nucleic acid sequences or the proportion of amino acidmatches between two amino acid sequences. When sequence homology isexpressed as a percentage, e.g., 50%, the percentage denotes theproportion of matches over the length of sequence from a desiredsequence (e.g., SEQ. ID NO: 1) that is compared to some other sequence.Gaps (in either of the two sequences) are permitted to maximizematching; gap lengths of 15 bases or less are usually used, 6 bases orless are used more frequently, with 2 bases or less used even morefrequently. The term “sequence identity” means that sequences areidentical (i.e., on a nucleotide-by-nucleotide basis for nucleic acidsor amino acid-by-amino acid basis for polypeptides) over a window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the comparison window,determining the number of positions at which the identical amino acidsoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the comparison window, and multiplying the result by 100 toyield the percentage of sequence identity. Methods to calculate sequenceidentity are known to those of skill in the art.

2. Biosensors

Provided are biosensors, compositions comprising biosensors, and methodsof using biosensors in living cells and organisms. The biosensors areable to be selectively targeted to certain regions or structures withina cell. The biosensors may provide a signal when the biosensor istargeted and/or in response to a property of the cell or organism suchas membrane potential, ion concentration or enzyme activity.

In general, the biosensors comprise at least two components; (1) aselectivity component capable of interacting with a target molecule ofinterest and a (2) reporter molecule that produces a detectable changein signal upon interaction of the selectivity component with the targetmolecule. The reporter molecule may be covalently linked to theselectivity component, or it may be able to noncovalently interact withthe selectivity component.

In various embodiments, the reporter molecule is responsive toenvironmental changes, including for example, pH sensitive molecules,restriction sensitive molecules, polarity sensitive molecules, andmobility sensitive molecules. The reporter molecule may be eitherfluorescent or chemiluminescent. In certain embodiments, the reportermolecule may interact with the selectivity component proximal to aregion that binds to the target molecule. In an exemplary embodiment,the reporter molecule is covalently attached to the selectivitycomponent proximal to a region that binds to the target molecule,optionally through an engineered reactive site. The biosensor mayrespond to changes in the concentration of the target molecule and maybe useful for monitoring the concentration of a target molecule overtime.

In certain embodiments, the biosensor may comprise two or more reportermolecules, which may be the same or different reporter molecules. Thereporter molecule may be detectable by a variety of methods, including,for example, a fluorescent spectrometer, filter fluorometer, microarrayreader, optical fiber sensor reader, epifluorescence microscope,confocal laser scanning microscope, two photon excitation microscope, ora flow cytometer.

In certain embodiments, methods for preparing the biosensors includegenerating selectivity components with an engineered reporter moleculebinding site using biological selection methods. The reporter moleculebinding site may be engineered to customize any of a number ofproperties, for example, for optimal binding affinity to the reportermolecule, to enhance or otherwise change or tune the signal from thereporter molecule when it binds the selectivity component, to provide areactive site in the reporter molecule binding site so that the reportermolecule may covalently associate with the selectivity component uponbinding in the binding site, or to modulate or perturb the activity ofthe selectivity component when the reporter molecule binds to it.

Accordingly, in certain embodiments, methods for generating a biosensormay comprise producing the selectivity component by genetic selection,genetic engineering or a combination of genetic selection and geneticengineering, so as to produce an engineered selectivity component.Methods for producing the engineered selectivity components aredescribed further below.

In other embodiments, particularly where the biosensor is produced froman endogenous source rather than expressed in the cell or tissue to beanalyzed, the biosensor may further comprise a chemical handle. Thechemical handle may be used to facilitate isolation, immobilization,identification, or detection of the biosensors and/or which increasesthe solubility of the biosensors.

In certain embodiments, the biosensor may be immobilized onto asubstrate surface, including, for example, substrates such as silicon,silica, quartz, glass, controlled pore glass, carbon, alumina, titania,tantalum oxide, germanium, silicon nitride, zeolites, gallium arsenide,gold, platinum, aluminum, copper, titanium, alloys, polystyrene,poly(tetra)fluoroethylene (PTFE), polyvinylidenedifluoride,polycarbonate, polymethylmethacrylate, polyvinylethylene,polyethyleneimine, poly(etherether)ketone, polyoxymethylene (POM),polyvinylphenol, polylactides, polymethacrylimide (PMI),polyalkenesulfone (PAS), polypropylethylene, polyethylene,polyhydroxyethylmethacrylate (HEMA), polydimethylsiloxane,polyacrylamide, polyimide, and block-copolymers. Such substrates may bein the form of beads, chips, plates, slides, strips, sheets, films,blocks, plugs, medical devices, surgical instruments, diagnosticinstruments, drug delivery devices, prosthetic implants, and otherstructures.

In another embodiment, the application provides a composition comprisingone or more biosensors. The composition may comprise a pharmaceuticallyacceptable carrier. The biosensors of the composition may be specificfor different target molecules, and may be associated with the same ordifferent reporter molecules.

In another embodiment, two or more biosensors may be immobilized onto asubstrate at spatially addressable locations. The biosensors may bespecific for different target molecules and may be associated with thesame or different reporter molecules.

In another aspect, the application provides a method for detecting atleast one target molecule comprising providing at least one biosensorcomprising a selectivity component and a reporter molecule and detectingthe signal of the reporter molecule, wherein interaction of thebiosensor with the target molecule produces a detectable change in thesignal of the reporter molecule. In various other aspects, thebiosensors of the invention may be used for the detection ofenvironmental pollutants, hazardous substances, food contaminants, andbiological and/or chemical warfare agents.

In various embodiments, the biosensors of the invention may be used todetect target molecules, including, for example, cells, microorganisms(bacteria, fungi and viruses), polypeptides, nucleic acids, hormones,cytokines, drug molecules, carbohydrates, pesticides, dyes, amino acids,small organic molecules and small inorganic molecules.

Biosensors may be used for the detection of target molecules both invivo and in vitro. In certain embodiments, the biosensor may be injectedor implanted into a patient and the signal of the reporter molecule isdetected externally. In one exemplary embodiment, the biosensors of theapplication may be used for the detection of intracellular targets. Inanother exemplary embodiment, the biosensors of the application may beattached to a fiber optic probe to facilitate position of the biosensorwithin a sample and readout from the biosensor through the opticalfiber.

In still other embodiments, the biosensor may be expressed directly intothe cell, tissue or subject to be analyzed. Using molecular biologymethods, a vector comprising at least a gene encoding a selectivitycomponent is constructed and inserted into the host, resulting inexpression of the selectivity component, as described in more detailbelow.

Various, more detailed embodiments of and methods for producing theselectivity component and reporter molecule components are also furtherdescribed below.

3. Selectivity Components

The selectivity component may be any molecule which is capable ofselectively interacting with a desired target molecule, including, forexample, cells, microorganisms (such as bacteria, fungi and viruses),polypeptides, nucleic acids (such as oligonucleotides, cDNA molecules orgenomic DNA fragments), hormones, cytokines, drug molecules,carbohydrates, pesticides, dyes, amino acids, or small organic orinorganic molecules.

Exemplary target molecules include, for example, molecules involved intissue differentiation and/or growth, cellular communication, celldivision, cell motility, and other cellular functions that take placewithin or between cells, including regulatory molecules such as growthfactors, cytokines, morphogenetic factors, neurotransmitters, and thelike. In certain embodiments, target molecules may be bone morphogenicprotein, insulin-like growth factor (IGF), and/or members of thehedgehog and Wnt polypeptide families.

Exemplary selectivity components include, for example, pathway andnetwork proteins (for example, enzymes such as kinases or phosphatases),antibody fragments, non-antibody receptor molecules, aptamers, templateimprinted materials, and organic or inorganic binding elements.Selectivity components having limited crossreactivity are generallypreferred.

3.A. Exemplary Polypeptide Selectivity Components

In certain embodiments, the selectivity component may be an antibody oran antibody fragment. For example, selectivity components may bemonoclonal antibodies, or derivatives or analogs thereof, includingwithout limitation: Fv fragments, single chain Fv (scFv) fragments, Fab′fragments, F(ab′)₂ fragments, single domain antibodies, camelizedantibodies and antibody fragments, humanized antibodies and antibodyfragments, and multivalent versions of the foregoing; multivalentselectivity components including without limitation: monospecific orbispecific antibodies, such as disulfide stabilized Fv fragments, scFvtandems ((scFv)₂ fragments), diabodies, tribodies or tetrabodies, whichtypically are covalently linked or otherwise stabilized (i.e., leucinezipper or helix stabilized) scFv fragments; receptor molecules whichnaturally interact with a desired target molecule.

In one embodiment, the selectivity component may be an antibody.Preparation of antibodies may be accomplished by any number ofwell-known methods for generating monoclonal antibodies. These methodstypically include the step of immunization of animals, typically mice;with a desired immunogen (e.g., a desired target molecule-or fragmentthereof). Once the mice have been immunized, and preferably boosted oneor more times with the desired immunogen(s), monoclonalantibody-producing hybridomas may be prepared and screened according towell known methods (see, for example, Kuby, Janis, IMMUNOLOGY, ThirdEdition, pp. 131-139, W.H. Freeman & Co. (1997), for a general overviewof monoclonal antibody production, that portion of which is incorporatedherein by reference).

Over the past several decades, antibody production has become extremelyrobust. In vitro methods that combine antibody recognition and phagedisplay techniques allow one to amplify and select antibodies with veryspecific binding capabilities. See, for example, Holt, L. J. et al.,“The Use of Recombinant Antibodies in Proteomics,” Current Opinion inBiotechnology 2000, 11:445-449, incorporated herein by reference. Thesemethods typically are much less cumbersome than preparation ofhybridomas by traditional monoclonal antibody preparation methods.Binding epitopes may range in size from small organic compounds such asbromo uridine and phosphotyrosine to oligopeptides on the order of 7-9amino acids in length.

In another embodiment, the selectivity component may be an antibodyfragment. Preparation of Antibody Fragments May be Accomplished by anyNumber of Well-Known methods. In one embodiment, phage displaytechnology may be used to generate antibody fragment selectivitycomponents that are specific for a desired target molecule, including,for example, Fab fragments, Fv's with an engineered intermoleculardisulfide bond to stabilize the V_(H)-V_(L) pair, scFvs, or diabodyfragments.

In certain embodiments, the selectivity component comprises apolypeptide sequence having at least about 85%, at least about 90%, atleast about 95%, about 96%, about 97%, about 98%, about 99% or about100% sequence identity to the polypeptide sequence of SEQ ID NO: 2 (FIG.1B). Vectors to produce the selectivity component may be prepared asdescribed below with the nucleic acid encoding the polypeptide of SEQ IDNO:2 and its homologs (for example, SEQ ID NO: 1 in FIG. 1A), and usedto transfect host cells as described further below.

As an example, production of scFv antibody fragments using phage displayis described below. However, scFv antibody fragments for use in theselectivity components may be generated by any method known in the artfor doing so, including genetic selection methods from a library ofyeast cells (see Boder and Wittrup (2000) Meth. Enzymol. 328:430-33;Boder, et al. (2000) Proc. Natl. Acad. Sci USA 97:10701-5; and Swers, etal. (2004) Nucl. Acids. Res. 32:e36).

For phage display, an immune response to a selected immunogen iselicited in an animal (such as a mouse, rabbit, goat or other animal)and the response is boosted to expand the immunogen-specific B-cellpopulation. Messenger RNA is isolated from those B-cells, or optionallya monoclonal or polyclonal hybridoma population. The mRNA isreverse-transcribed by known methods using either a poly-A primer ormurine immunoglobulin-specific primer(s), typically specific tosequences adjacent to the desired V_(H) and V_(L) chains, to yield cDNA.The desired V_(H) and A chains are amplified by polymerase chainreaction (PCR) typically using V_(H) and A specific primer sets, and areligated together, separated by a linker. V_(H) and V_(L) specific primersets are commercially available, for instance from Stratagene, Inc. ofLa Jolla, Calif. Assembled V_(H)-linker-V_(L) product (encoding an scFvfragment) is selected for and amplified by PCR. Restriction sites areintroduced into the ends of the V_(H)-linker-V_(L) product by PCR withprimers including restriction sites and the scFv fragment is insertedinto a suitable expression vector (typically a plasmid) for phagedisplay. Other fragments, such as an Fab′ fragment, may be cloned intophage display vectors for surface expression on phage particles. Thephage may be any phage, such as lambda, but typically is a filamentousphage, such as fd and M 13, typically M13.

In phage display vectors, the V_(H)-linker-V_(L) sequence is cloned intoa phage surface protein (for M 13, the surface proteins g3p (pHI) org8p, most typically g3p). Phage display systems also include phagemidsystems, which are based on a phagemid plasmid vector containing thephage surface protein genes (for example, g3p and g8p of M13) and thephage origin of replication. To produce phage particles, cellscontaining the phagemid are rescued with helper phage providing theremaining proteins needed for the generation of phage. Only the phagemidvector is packaged in the resulting phage particles because replicationof the phagemid is grossly favored over replication of the helper phageDNA. Phagemid packaging systems for production of antibodies arecommercially available. One example of a commercially available phagemidpackaging system that also permits production of soluble ScFv fragmentsin bacteria cells is the Recombinant Phage Antibody System (R.PAS),commercially available from Amersham Pharmacia Biotech, Inc. ofPiscataway, N.J. and the pSKAN Phagemid Display System, commerciallyavailable from MoBiTec, LLC of Marco Island, Fla. Phage display systems,their construction and screening methods are described in detail in,among others, U.S. Pat. Nos. 5,702,892, 5,750,373, 5,821,047 and6,127,132, each of which are incorporated herein by reference in theirentirety.

Typically, once phage are produced that display a desired antibodyfragment, epitope specific phage are selected by their affinity for thedesired immunogen and, optionally, their lack be used for physicallyseparating immunogen-binding phage from non-binding phage. Typically theimmunogen is fixed to a surface and the phage are contacted with thesurface. Non-binding phage are washed away while binding phage remainbound. Bound phage are later eluted and are used to re-infect cells toamplify the selected species. A number of rounds of affinity selectiontypically are used, often increasingly higher stringency washes, toamplify immunogen binding phage of increasing affinity. Negativeselection techniques also may be used to select for lack of binding to adesired target. In that case, un-bound (washed) phage are amplified.

Although it is preferred to use spleen cells and/or B-lymphocytes fromanimals preimmunized with a desired immunogen as a source of cDNA fromwhich the sequences of the V_(H) and V_(L) chains are amplified byRT-PCR, naive (un-immunized with the target immunogen) splenocytesand/or B-cells may be used as a source of cDNA to produce a polyclonalset of V_(H) and V_(L) chains that are selected in vitro by affinity,typically by the above-described phage display (phagemid) method. Whennaive B-cells are used, during affinity selection, the washing of thefirst selection step typically is of very high stringency so as to avoidloss of any single clone that may be present in very low copy number inthe polyclonal phage library. By this naive method, B-cells may beobtained from any polyclonal source, B-cell or splenocyte cDNA librariesalso are a source of cDNA from which the V_(H) and V_(L) chains may beamplified. For example, suitable murine and human B-cell, lymphocyte andsplenocyte cDNA libraries are commercially available from Stratagene,Inc. and from Clontech Laboratories, Inc. of Palo Alto, Calif. Phagemidantibody libraries and related screening services are providedcommercially by Cambridge Antibody Technology of the U.K. or MorphoSysUSA, Inc., of Charlotte, N.C.

The selectivity components do not have to originate from biologicalsources, such as from naive or immunized immune cells of animals orhumans. The selectivity components may be screened from a combinatoriallibrary of synthetic peptides. One such method is described in U.S. Pat.No. 5,948,635, incorporated herein by reference, which described theproduction of phagemid libraries having random amino acid insertions inthe pIII gene of M13. These phage may be clonally amplified by affinityselection as described above.

Panning in a culture dish or flask is one way to physically separatebinding phage from non-binding phage. Panning may be carried out in 96well plates in which desired immunogen structures have been immobilized.Functionalized 96 well plates, typically used as ELISA plates, may bepurchased from Pierce of Rockwell, Ill. Polypeptides immunogens may besynthesized directly on NH₂ or COOH functionalized plates in anN-terminal to C-terminal direction. Other affinity methods for isolatingphage having a desired specificity include affixing the immunogen tobeads. The beads may be placed in a column and phage may be bound to thecolumn, washed and eluted according to standard procedures.Alternatively, the beads may be magnetic so as to permit magneticseparation of the binding particles from the non-binding particles. Theimmunogen also may be affixed to a porous membrane or matrix, permittingeasy washing and elution of the binding phage.

In certain embodiments, it may be desirable to increase the specificityof the selectivity component for a given target molecule or reportermolecule using a negative selection step in the affinity selectionprocess. For example, selectivity component displaying phage may becontacted with a surface functionalized with immunogens distinct fromthe target molecule or reporter molecule. Phage are washed from thesurface and non-binding phage are grown to clonally expand thepopulation of non-binding phage thereby de-selecting phage that are notspecific for the desired target molecule. In certain embodiments, randomsynthetic peptides may be used in the negative selection step. In otherembodiments, one or more immunogens having structural similarity to thetarget molecule or reporter molecule may be used in the negativeselection step. For example, for a target molecule comprising apolypeptide, structurally similar immunogens may be polypeptides havingconservative amino acid substitutions, including but not limited to theconservative substitution groups such as: (i) a charged group,consisting of Glu and Asp, Lys, Arg and His, (ii) a positively-chargedgroup, consisting of Lys, Arg and His, (iii) a negatively-charged group,consisting of Glu and Asp, (iv) an aromatic group, consisting of Phe,Tyr and Trp, (v) a nitrogen ring group, consisting of His and Trp, (vi)a large aliphatic nonpolar group, consisting of Val, Leu and Ile, (vii)a slightly polar group, consisting of Met and Cys, (viii) asmall-residue group, consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu,Gln and Pro, (ix) an aliphatic group consisting of Val, Leu, Ile, Metand Cys, and (x) a small hydroxyl group consisting of Ser and Thr.Conservative substitutions also may be determined by one or moremethods, such as those used by the BLAST (Basic Local Alignment SearchTool) algorithm, such as a BLOSUM Substitution Scoring Matrix; such asthe BLOSUM 62 matrix, and the like. A functional way to define commonproperties between individual amino acids is to analyze the normalizedfrequencies of amino acid changes between corresponding proteins ofhomologous

Screening of selectivity components will best be accomplished by highthroughput parallel selection, as described in Holt et al.Alternatively, high throughput parallel selection may be conducted bycommercial entities, such as by Cambridge Antibody Technologies orMorphoSys USA, Inc.

Alternatively, selection of a desired selectivity component-displayingphage may be carried out using the following method:

Step 1: Affinity purify phage under low stringency conditions for theirability to bind to an immunogen fixed to a solid support (for instance,beads in a column).

Step 2: Elute the bound phage and grow the eluted phage. Steps 1 and 2may be repeated with more stringent washes in Step 1.

Step 3: Absorb the phage under moderate stringency with a given proteinmixture digested with a proteolytic agent of interest. Wash away theunbound phage with a moderately stringent wash and grow the washedphage. Step 3 may be repeated with less stringent washes.

Step 4: Affinity purify phage under high stringency for their ability tobind to the immunogen fixed to a solid support. Elute the bound phageand grow the eluted phage.

Step 5: Plate the phage to select single plaques. Independently growphage selected from each plaque and confirm the specificity to thedesired immunogen.

This is a general-guideline for the clonal expansion ofimmunogen-specific selectivity components. Additional steps of varyingstringency may be added at any stage to optimize the selection process,or steps may be omitted or re-ordered. One or more steps may be addedwhere the phage population is selected for its inability to bind toother immunogens by absorption of the phage population with those otherimmunogens and amplification of the unbound phage population. That stepmay be performed at any stage, but typically would be performed afterstep 4.

In certain embodiments, it may be desirable to mutate the binding regionof the selectivity component and select for selectivity components withsuperior binding characteristics as compared to the un-mutatedselectivity component. This may be accomplished by any standardmutagenesis technique, such as by PCR with Taq polymerase underconditions that cause errors. In such a case, the PCR:primers could beused to amplify scFv-encoding sequences of phagemid plasmids underconditions that would cause mutations. The PCR product may then becloned into a phagemid vector and screened for the desired specificity,as described above.

In other embodiments, the selectivity components may be modified to makethem more resistant to cleavage by proteases. For example, the stabilityof the selectivity components of the present invention that comprisepolypeptides may be increased by substituting one or more of thenaturally occurring amino acids in the (L) configuration with D-aminoacids. In various embodiments, at least 1%, 5%, 10%, 20%, 50%, 80%, 90%or 100% of the amino acid residues of the selectivity components may beof the D configuration. The switch from L to D amino acids neutralizesthe digestion capabilities of many of the ubiquitous peptidases found inthe digestive tract. Alternatively, enhanced stability of theselectivity components of the invention may be achieved by theintroduction of modifications of the traditional peptide linkages. Forexample, the-introduction of a cyclic ring within the polypeptidebackbone may confer enhanced stability in order to circumvent the effectof many proteolytic enzymes known to digest polypeptides in the stomachor other digestive organs and in serum. In still other embodiments,enhanced stability of the selectivity components may be achieved byintercalating one or more dextrorotatory amino acids (such as,dextrorotatory phenylalanine or dextrorotatory tryptophan) between theamino acids of the selectivity component. In exemplary embodiments, suchmodifications increase the protease resistance of the selectivitycomponents without affecting their activity or specificity ofinteraction with a desired target molecule or reporter molecule.

In certain embodiments, the antibodies or variants thereof, may bemodified to make them less immunogenic when administered to a subject.For example, if the subject is human, the antibody may be “humanized”;where the complimentarily determining region(s) of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones, P. et al. (1986), Nature 321, 522-525,Tempest et al. (1991) Biotechnology 9, 266-273, and U.S. Pat. No.6,407,213. Also, transgenic mice, or other mammals, may be used toexpress humanized antibodies. Such humanization may be partial orcomplete.

In another embodiment, the selectivity component is a Fab fragment. Fabantibody fragments may be obtained by proteolysis of an immunoglobulinmolecule using the protease papain. Papain digestion yields twoidentical antigen-binding fragments, termed “Fab fragments”, each with asingle antigen-binding site, and a residual “Fc fragment”. In anexemplary embodiment, papain is first activated by reducing thesulfhydryl group in the active site with cysteine, mercaptoethanol ordithiothreitol. Heavy metals in the stock enzyme may be removed bychelation with EDTA (2 mM) to ensure maximum enzyme activity. Enzyme andsubstrate' are normally mixed together in the ratio of 1:100 by weight.After incubation, the reaction can be stopped by irreversible alkylationof the thiol group with iodoacetamide or simply by dialysis. Thecompleteness of the digestion should be monitored by SDS-PAGE and thevarious fractions separated by protein A-Sepharose or ion exchangechromatography.

In still another embodiment, the selectivity component is a F(ab′)₂fragment. F(ab′)₂ antibody fragments may be prepared from IgG moleculesusing limited proteolysis with the enzyme pepsin. Exemplary conditionsfor pepsin proteolysis are 100 times antibody excess w/w in acetatebuffer at pH 4.5 and 37° C. Pepsin treatment of intact immunoglobulinmolecules yields an F(ab′)₂ fragment that has two antigen-combiningsites and is still capable of crosslinking antigen. Fab′ antibodyfragments may be obtained by reducing F(ab′)₂ fragments using2-mercaptoethylamine. The Fab′ fragments may be separated from unsplitF(ab′)₂ fragments and concentrated by application to a Sephadex G-25column (MT=46,000-58,000). In other embodiments, the selectivitycomponent may be a non-antibody receptor molecule, including, forexample, receptors which naturally recognize a desired target molecule,receptors which have been modified to increase their specificity ofinteraction with a target molecule, receptor molecules which have beenmodified to interact with a desired target molecule not naturallyrecognized by the receptor, and fragments of such receptor molecules(see, e.g., Skerra, J. Molecular Recognition 13: 167-187 (2000)).

In other embodiments, the selectivity component may be a network orpathway protein such as an enzyme, for example, a phosphatase or kinase.Such proteins may be mutated to create a binding site for a reporterand/or target molecule. For example, a method of creating a biosensorfrom network and pathway proteins in cells and tissues may comprisemutating a specific region on the selected protein to create a bindingsite for a reporter or target molecule. The region selected for mutationmay be randomly or partially randomly mutated by creating mutations inselected regions of the gene that codes for the protein that is to beconverted into a selectivity component. The gene with the mutatedregion(s) may be incorporated by transfection into a system capable ofexpressing the protein in a way that allows reporter molecule (or targetmolecule) binding and fluorescence sensitivity to the activity (if areporter molecule) to be assayed. For example, the DNA with the mutatedregion may be transfected into yeast cells that are able to express manycopies of the mutated protein molecules on the cell surface (see Boderand Wittrup (2000) Meth. Enzymol. 328:430-33; Boder, et al. (2000) Proc.Natl. Acad. Sci USA 97:10701-5; and Swers, et al. (2004) Nucl. Acids.Res. 32:e36). By isolating and identifying by selection methods thegenetic sequence of the particular protein within the mutated populationthat functions optimally as a selectivity component. For example,reporter molecule binding mutants may be detected and selected usingmagnetic bead separation and by flow cytometry or image cytometry.Mutants that show a particular fluorescence signal change from boundreporter molecule in response to protein activity changes may bedetected and isolated. In the case of engineering a reporter moleculebinding site that is reactive, a reactive group may be engineered intothe site (such as a thiol) and ability to covalently bind the reportermolecule may be assayed. A biosensor can then be produced by combiningthe reporter molecule with the optimized selectivity componentcontaining the engineered site.

In other embodiments, a library of mutants is generated from adegenerate oligonucleotide sequence. There are many ways by which thelibrary may be generated from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence may be carried out inan automatic DNA synthesizer, and the synthetic genes may then beligated into an appropriate vector for expression. One purpose of adegenerate set of genes is to provide, in one mixture, all of thesequences encoding the desired set of potential protein sequences. Thesynthesis of degenerate oligonucleotides is well known in the art (seefor example, Narang, S A (1983) Tetrahedron 39:3; Itakura et al., (1981)Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AGWalton, Amsterdam: Elsevier pp. 273-289; Itakura et al., (1984) Annu.Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike etal., (1983) Nucleic Acid Res. 11:477). Such techniques have beenemployed in the directed evolution of other proteins (see, for example,Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) Proc.Natl. Acad. Sci. USA 89:2429-2433; Devlin et al., (1990) Science 249:404-406; Cwirla et al., (1990) Proc. Natl. Acad. Sci. USA 87: 6378-6382;as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis may be utilized to generate acombinatorial library. For example, mutants may be generated andisolated from a library by screening using, for example, alaninescanning mutagenesis and the like (Ruf et al., (1994) Biochemistry33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balintet al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J.Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem.268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838; andCunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell. Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis (Miller et al., (1992) A Short Coursein Bacterial Genetics, CSHL Press, Cold Spring Harbor, N.Y.; and Greeneret al., (1994) Strategies in Mol Biol 7:32-34). Linker scanningmutagenesis, particularly in a combinatorial setting, is an attractivemethod for identifying selectivity components.

3.B. Exemplary Polynucleotide Selectivity Components

In still other embodiments, the selectivity component may be an aptamer.Aptamers are oligonucleotides that are selected to bind specifically toa desired molecular structure. Aptamers typically are the products of anaffinity selection process similar to the affinity selection of phagedisplay (also known as in vitro molecular evolution). The processinvolves performing several tandem iterations of affinity separation,e.g., using a solid support to which the desired immunogen is bound,followed by polymerase chain reaction (PCR) to amplify nucleic acidsthat bound to the immunogens. Each round of affinity separation thusenriches the nucleic acid population for molecules that successfullybind the desired immunogen. In this manner, a random pool of nucleicacids may be “educated” to yield aptamers that specifically bind targetmolecules. Aptamers typically are RNA, but may be DNA or analogs orderivatives thereof, such as, without limitation, peptide nucleic acidsand phosphorothioate nucleic acids.

In exemplary embodiments, nucleic acid ligands, or aptamers, may beprepared using the “SELEX” methodology which involves selection ofnucleic acid ligands which interact with a target in a desirable mannercombined with amplification of those selected nucleic acids. The SELEXprocess, is described in U.S. Pat. Nos. 5,475,096 and 5,270,163 and PCTApplication No. WO 91/19813. These references, each specificallyincorporated herein by reference, are collectively called the SELEXPatents.

The SELEX process provides a class of products which are nucleic acidmolecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired target compound ormolecule. In various embodiments, target molecules may be, for example,proteins, carbohydrates, peptidoglycans or small molecules. SELEXmethodology can also be used to target biological structures, such ascell surfaces or viruses, through specific interaction with a moleculethat is an integral part of that biological structure.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A candidate mixture of nucleic acids of differing sequence, isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either, (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the target, or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one. in four) oronly partially randomized (e.g., the probability of finding a base atany location can be selected at any level between 0 and 100 percent).

2) The candidate mixture is contacted with the selected target underconditions favorable for binding between the target and members of thecandidate mixture. Under these circumstances, the interaction betweenthe target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning.

4) Those nucleic acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newlyformed candidate mixture contains fewer and fewer unique sequences, andthe average degree of affinity of the nucleic-acids to the target willgenerally increase. The SELEX-process ultimately may yield a candidatemixture containing one or a small number of unique nucleic acidsrepresenting those nucleic acids from the original candidate mixturehaving the highest affinity to the target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. Pat. No. 5,707,796 describes the use ofthe SELEX process in conjunction with gel electrophoresis to selectnucleic acid molecules with specific structural characteristics, such asbent DNA. U.S. Pat. No. 5,580,737 describes a method for identifyinghighly specific nucleic acid ligands able to discriminate betweenclosely related molecules, termed CounterSELEX. U.S. Pat. No. 5,567,588describes a SELEX-based method which achieves highly efficientpartitioning between oligonucleotides having high and low affinity for atarget molecule. U.S. Pat. Nos. 5,496,938 and 5,683,867 describe methodsfor obtaining improved nucleic acid ligands after SELEX has beenperformed.

In certain-embodiments, nucleic acid ligands as described herein maycomprise modifications that increase their stability, including, forexample, modifications that provide increased resistance to degradationby enzymes such as endonucleases and exonucleases, and/or modificationsthat enhance or mediate the delivery of the nucleic acid ligand (see,e.g., U.S. Pat. Nos. 5,660,985 and 5,637,459). Examples of suchmodifications include chemical substitutions at the ribose and/orphosphate and/or base positions. In various embodiments, modificationsof the nucleic acid ligands may include, but are not limited to, thosewhich provide other chemical groups that incorporate additional charge,polarizability, hydrophobicity, hydrogen bonding, electrostaticinteraction, and fluxionality to the nucleic acid ligand bases or to thenucleic acid ligand as a whole. Such modifications include, but are notlimited to, 2′-position sugar modifications, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atexocyclic amines, substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil; backbone modifications, phosphorothioate or alkylphosphate modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications may also include 3′ and 5′ modifications such as capping.In exemplary embodiments, the nucleic acid ligands are RNA moleculesthat are 2′-fluoro (2′-F) modified on the sugar moiety of pyrimidineresidues.

3.C. Other Exemplary Selectivity Components

In other embodiments, the selectivity components may be templateimprinted material. Template imprinted materials are structures whichhave an outer sugar layer and an underlying plasma-deposited layer. Theouter sugar layer contains indentations or imprints which arecomplementary in shape to a desired target molecule or template so as toallow specific interaction between the template imprinted structure andthe target molecule to which it is complementary. Template imprintingcan be utilized on the surface of a variety of structures, including,for example, medical prostheses (such as artificial heart valves,artificial limb joints, contact lenses and stents), microchips(preferably silicon-based microchips) and components of diagnosticequipment designed to detect specific microorganisms, such as viruses orbacteria. Template-imprinted materials are discussed in U.S. Pat. No.6,131,580, which is hereby incorporated by reference in its entirety.

3.D. Modification of Selectivity Components for Incorporation intoBiosensors and Exemplary Embodiments Wherein the Selectivity Componentis Produced Independently of the Cell or Tissue to be Analyzed

In certain embodiments, a selectivity component of the invention maycontain a chemical handle which facilitates its isolation,immobilization, identification, or detection and/or which increases itssolubility. In various embodiments, chemical handles may be apolypeptide, a polynucleotide, a carbohydrate, a polymer, or a chemicalmoiety and combinations or variants thereof. In certain embodiments,exemplary chemical handles, include, for example, glutathioneS-transferase (GST); protein A, protein G, calmodulin-binding peptide,thioredoxin, maltose binding protein, HA, myc, poly arginine, poly H is,poly His-Asp or FLAG tags. Additional exemplary chemical handles includepolypeptides that alter protein localization in vivo, such as signalpeptides, type III secretion system-targeting peptides, transcytosisdomains, nuclear localization signals, etc.

In various embodiments, a selectivity component of the invention maycomprise one or more chemical handles, including multiple copies of thesame chemical handle or two or more different chemical handles. It isalso within the scope of the invention to include a linker (such as apolypeptide sequence or a chemical moiety) between a selectivitycomponent of the invention and the chemical handle in order tofacilitate construction of the molecule or to optimize its structuralconstraints.

In another embodiment, a selectivity component of the invention may bemodified so that its rate of traversing the cellular membrane isincreased. For example, the selectivity component may be attached to apeptide which promotes “transcytosis,” e.g., uptake of a polypeptide bycells. The peptide may be a portion of the HIV transactivator (TAT)protein, such as the fragment corresponding to residues 37-62 or 48-60of TAT, portions which have been observed to be rapidly taken up by acell in vitro (Green and Loewenstein, (1989) Cell 55:1179-1188).Alternatively, the internalizing peptide may be derived from theDrosophila antennapedia protein, or homologs thereof. The 60 amino acidlong homeodomain of the homeo-protein antennapedia has been demonstratedto translocate through biological membranes and can facilitate thetranslocation of heterologous polypeptides to which it-is coupled. Thus,selectivity components may be fused to a peptide consisting of aboutamino acids 42-58 of Drosophila antennapedia or shorter fragments fortranscytosis (Derossi et al. (1996) J Biol Chem 271:18188-18193; Derossiet al. (1994) J Biol Chem 269:10444-10450; and Perez et al. (1992) JCell Sci 1.02:717-722). The transcytosis polypeptide may also be anon-naturally-occurring membrane-translocating sequence (MTS), such asthe peptide sequences disclosed in U.S. Pat. No. 6,248,558.

In still other embodiments, the selectivity component may comprise afusion protein of any of the above-described polypeptide selectivitycomponents containing at least one domain which increases its solubilityand/or facilitates its purification, identification, detection,targeting and/or delivery. Exemplary domains, include, for example,glutathione S-transferase (GST), protein A, protein G,calmodulin-binding peptide, thioredoxin, maltose binding protein, HA,myc, poly arginine, poly His, poly His-Asp or FLAG fusion proteins andtags. Additional exemplary domains include domains that alter proteinlocalization in vivo, such as signal peptides, type III secretionsystem-targeting peptides, transcytosis domains, nuclear localizationsignals, and targeting moieties, i.e. proteins specific for a targetmolecule, etc. In various embodiments, a polypeptide of the inventionmay comprise one or more heterologous fusions. Polypeptides may containmultiple copies of the same fusion domain or may contain fusions to twoor more different domains. The fusions may occur at the N-terminus ofthe polypeptide, at the C-terminus of the polypeptide, or at both the N-and C-terminus of the polypeptide. Linker sequences between apolypeptide of the invention and the fusion domain may be included inorder to facilitate construction of the fusion protein or to optimizeprotein expression or structural constraints of the fusion protein.

In exemplary embodiments, the dissociation constant of the selectivitycomponent for a target molecule is optimized to allow real timemonitoring of the presence and/or concentration of the analyte in agiven patient, sample, or environment.

The selectivity components (for example, phage, antibodies, antibodyfragments, aptamers, etc.) may be affixed to a suitable substrate by anumber of known methods. Typically the surface of the substrate isfunctionalized in some manner, so that a crosslinking compound orcompounds may covalently link the selectivity component to thesubstrate. For example, a substrate functionalized with carboxyl groupsmay be linked to free amines in the selectivity components using EDC orby other common chemistries, such as by linking withN-hydroxysuccinimide. A variety of crosslinking chemistries arecommercially available, for instance, from Pierce of Rockford, Ill.

For attachment of the sensor units to surfaces there are a number oftraditional attachment technologies. For example, activated carboxylgroups on the substrate will link the sensor units to the substrate via—NH₂ groups on the selectivity component of the biosensor. The substrateof the array may be either organic or inorganic, biological ornon-biological, or any combination of these materials. Numerousmaterials are suitable for use as a substrate for the sensor units ofthe invention. For instance, the substrate of the invention sensors cancomprise a material selected from a group consisting of silicon, silica,quartz, glass, controlled pore glass, carbon, alumina, titania, tantalumoxide, germanium, silicon nitride, zeolites, and gallium arsenide. Manymetals such as gold, platinum, aluminum, copper, titanium, and theiralloys are also options for substrates of the array. In addition, manyceramics and polymers may also be used as substrates. Polymers which maybe used as substrates include, but are not limited to, the following:polystyrene; poly(tetra)fluoroethylene (PTFE); polyvinylidenedifluoride;polycarbonate; polymethylmethacrylate; polyvinylethylene;polyethyleneimine; poly(etherether)ketone; polyoxymethylene (POM);polyvinylphenol; polylactides; polymethacrylimide (PMI);polyalkenesulfone (PAS); polypropylethylene, polyethylene;polyhydroxyethylmethacrylate (HEMA); polydimethylsiloxane;polyacrylamide; polyimide; and block copolymers. Preferred substratesfor the array include silicon, silica, glass, and polymers. Thesubstrate on which the sensors reside may also be a combination of anyof the aforementioned substrate materials.

A biosensor of the present invention may optionally further comprise acoating between the substrate and the bound biosensor molecule. Thiscoating may either be formed on the substrate or applied to thesubstrate. The substrate can be modified with a coating by usingthin-film technology based, for instance, on physical vapor deposition(PVD), plasma-enhanced chemical vapor deposition (PECVD), or thermalprocessing. Alternatively, plasma exposure can be used to directlyactivate or alter the substrate and create a coating. For instance,plasma etch procedures can be used to oxidize a polymeric surface (forexample, polystyrene or polyethylene to expose polar functionalitiessuch as hydroxyls, carboxylic acids, aldehydes and the like) which thenacts as a coating.

The coating may also comprise a composition selected from the groupconsisting of silicon, silicon oxide, titania, tantalum oxide, siliconnitride, silicon hydride, indium tin oxide, magnesium oxide, alumina,glass, hydroxylated surfaces, and polymers.

The substrate surface shall comprise molecules of formulaX(a)-R(b)-Y(c), wherein R is a spacer, X is a functional group thatbinds R to the surface, Y is a functional group for binding to thebiosensor, (a) is an integer from 0 to about 4, (b) is either 0 or 1,and (c) is an integer not equal to 0. Note that when both (a) and (b)are zero, the substrate surface comprises functional groups Y as wouldbe seen, for example, with polymeric substrates or coatings. When (a)and (b) are not equal to 0, then X(a)-R(b)-Y(c) describes, for example,monolayers such as a self assembled monolayers that form on a metalsurface. X(a)-R(b)-Y(c) may also describe such compounds as3-aminopropyltrimethoxysilane, wherein X is —Si(OMe)₃, R is —CH₂CH₂CH₂—,and Y is —NH₂. This compound is known to coat porous glass surfaces toform an aminopropyl derivative of the glass. Biochem. Biophys. Act.,1970, 212, 1; J. Chromatography, 1974, 97, 39.

Other definitions for F, X, and Y include the following. R optionallycomprises a linear or branched hydrocarbon chain from about 1 to about400 carbons long. The hydrocarbon chain may comprise an alkyl, aryl,alkenyl, alkynyl, cycloalkyl, alkaryl, aralkyl group, or any combinationthereof. If (a) and (c) are both equal to one, then R is typically analkyl chain from about 3 to about 30 carbons long. In a preferredembodiment, if (a) and (b) are both equal to one, then R is an alkyl,chain from about 8 to about 22 carbons long and is, optionally, astraight alkane. However, it is also contemplated that in an alternativeembodiment, R may readily comprise a linear or branched hydrocarbonchain from about 2 to about 400 carbons long and be interrupted by atleast one hetero atom. The interrupting hetero groups can include —O—,—CONH₂, —CONHCO—, —NH—, —CSNH—, —CO—, —CS—, —S—, —SO—, —(OCH₂CH₂)n-(where n=1-20), —(CF₂)n (where n=1-22), and the like. Alternatively, oneor more of the hydrogen moieties of R can be substituted with deuterium.In alternative embodiments, R may be more than about 400 carbons long.

X may be chosen as any group which affords chemisorption orphysisorption of the monolayer onto the surface of the substrate (or thecoating, if present). When the substrate or coating is a-metal or metalalloy, X, at least prior to incorporation into the monolayer, can in oneembodiment be chosen to be an asymmetrical or symmetrical disulfide,sulfide, diselenide, selenide, thiol, isonitrile, selenol, a trivalentphosphorus compound, isothiocyanate, isocyanate, xanthanate,thiocarbamate, a phosphine, an amine, thio acid or a dithio acid. Thisembodiment is especially preferred when a coating or substrate is usedthat is a noble metal such as gold, silver, or platinum.

If the substrate is a material such as silicon, silicon oxide, indiumtin oxide, magnesium oxide, alumina, quartz, glass, or silica, then, inone embodiment, the biosensor may comprise an X that, prior toincorporation into said monolayer, is a monohalosilane, dihalosilane,tihalosilane, trialkoxysilane, dialkoxysilane, or a monoalkoxysilane.Among these silanes, trichlorosilane and trialkoxysilane are exemplary.

In certain embodiments, the substrate is selected from the groupconsisting of silicon, silicon dioxide, indium tin oxide, alumina,glass, and titania; and X is selected from the group consisting of amonohalosilane, dihalosilane, tihalosilane, trichlorosilane,trialkoxysilane, dialkoxysilane, monoalkoxysilane, carboxylic acids, andphosphates.

In another embodiment, the substrate of the sensor is silicon and X isan olefin.

In still another embodiment, the coating (or the substrate if no coatingis present) is titania or tantalum oxide and X is a phosphate.

In other embodiments, the surface of the substrate (or coating thereon)is composed of a material such as titanium oxide; tantalum oxide, indiumtin oxide, magnesium oxido, or alumina where X is a carboxylic acid oralkylphosphoric acid. Alternatively, if the surface of the substrate (orcoating thereon) of the sensor is copper, then X may optionally. be ahydroxamic acid.

If the substrate used in the invention is a polymer, then in many casesa coating on the substrate such as a copper coating will be included inthe sensor. An appropriate functional group X for the coating would thenbe chosen for use in the sensor. In an alternative embodiment comprisinga polymer substrate, the surface of the polymer may be plasma modifiedto expose desirable surface functionalities for monolayer formation. Forinstance, EP 780423 describes the use of a monolayer molecule that hasan alkene X functionality on a plasma exposed surface. Still anotherpossibility for the invention sensor comprised of a polymer is that thesurface of the polymer on which the monolayer is formed isfunctionalized by copolymerization of appropriately functionalizedprecursor molecules.

Another possibility is that prior to incorporation into the monolayer, Xcan be a free radical-producing moiety. This functional group isespecially appropriate when the surface on which the monolayer is formedis a hydrogenated silicon surface. Possible free-radical producingmoieties include, but are not limited to, diacylperoxides, peroxides,and azo compounds. Alternatively, unsaturated moieties such asunsubstituted alkenes, alkynes, cyanocompounds and isonitrile compoundscan be used for —X, if the reaction with X is accompanied byultraviolet, infrared, visible, or microwave radiation.

In alternative embodiments, X may be a hydroxyl, carboxyl, vinyl,sulfonyl, phosphoryl, silicon hydride, or an amino group.

The component Y is a functional group responsible for binding a dyecontaining sensor onto the substrate. In one embodiment, the Y group iseither highly reactive (activated) towards the dye containing sensor oris easily converted into such an activated form. In certain embodiments,the coupling of Y with the selectivity component of the biosensor occursreadily under normal physiological conditions. The functional group Ymay either form a covalent linkage or a noncovalent linkage with theselectivity component of the biosensor. In other embodiments, thefunctional group Y forms a covalent linkage with the selectivitycomponent of the biosensor. It is understood that following theattachment of the selectivity component of the biosensor to Y, thechemical nature of Y may have changed. Upon attachment of the biosensor,Y may even have been removed from the organic linker.

In one embodiment of the sensor of the present invention, Y is afunctional group that is activated in situ. Possibilities for this typeof functional group include, but are not limited to, such simplemoieties such as a hydroxyl, carboxyl, amino, aldehyde, carbonyl,methyl, methylene, alkene, allyne, carbonate, aryliodide, or a vinylgroup. Appropriate modes of activation would be obvious to one skilledin the art. Alternatively, Y can comprise a functional group thatrequires photoactivation prior to becoming activated enough to trap theprotein capture agent.

In another embodiment, Y is a complex and highly reactive functionalmoiety that needs no in situ activation prior to reaction with theselectivity component of the biosensor. Such possibilities for Yinclude, but are not limited to, maleimide, N-hydroxysuccinimide (Wagneret al., Biophysical Journal, 1996, 70:2052-2066), nitrilotriacetic acid(U.S. Pat. No. 5,620,850), activated hydroxyl, haloacetyl, bromoacetyl,iodoacetyl, activated carboxyl, hydrazide, epoxy, aziridine,sulfonylchloride, trifluoromethyldiaziridine, pyridyldisulfide,N-acyl-imidazole, imidazolcearbatnate, vinylsulfone,succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenone,isothiocyanate, isocyanate, imidoester, fluorobenzene, and biotin.

In an alternative embodiment, the functional group Y is selected fromthe group of simple functional moieties. Possible Y functional groupsinclude, but are not limited to —OH, —NH₂, —COOH, —COOR, —RSR, —PO₄ ⁻³,—OSO₃ ⁻², —COONR₂, SOO⁻, —CN, —NR₂, and the like.

In another embodiment, one or more biosensor species may be bound todiscrete beads or microspheres. The microspheres typically are eithercarboxylated or avidin-modified so that proteins, such as antibodies,non-antibody receptors and variants and fragments thereof, may bereadily attached to the beads by standard chemistries. In an exemplaryembodiment, the selectivity components are scFv fragments. The scFvfragments may be bound to carboxylated beads by one of many linkingchemistries, such as, for example, EDC chemistry, or bound toavidin-coated beads by first biotinylating the scFv fragment by one ofmany common biotinylation chemistries, such as, for example, byconjugation with sulfo-NHS-LC-biotin.

In another embodiment, two or more biosensors are affixed to one or moresupports at discrete locations (that is, biosensors having a firstspecificity are affixed at a first spatial location, biosensors having asecond specificity are affixed at a second spatial location, etc.). Inone embodiment, the biosensors are affixed to a substrate in a tiledarray, with each biosensor represented in one or more positions in thetiled array. The spatial configuration of the substrate or substratesmay be varied so long as each biosensor species is bound at detectablydiscrete locations. The substrate and tiled biosensor pattern typicallyis planar, but may be any geometric configuration desired. For instance,the substrate may be a strip or cylindrical, as illustrated in U.S. Pat.No. 6,057,100. In exemplary embodiments, the substrate may be glass-orother silicic compositions; such as those used in the semiconductorindustry.

Fabrication of the substrate may be by one of many well-known processes.In various embodiments, the biosensors of the array may be associatedwith the same reporter molecule or may be associated with differentreporter molecules. Identification of a biosensor that interacts with atarget molecule may be based on the signal from the reporter molecule,the location of the biosensor on the array, or a combination thereof.Arrays may be used in association with both the in vitro and in vivoapplications of the invention.

In various embodiments, the arrays may comprise any of the biosensorsdescribed herein, including, for example, arrays of biosensors whereinthe selectivity components are polypeptides (including, antibodies andvariants or fragments thereof), polynucleotides (i.e., aptamers),template imprinted materials, organic binding elements, and inorganicbinding elements. The arrays may comprise one type of biosensor or amixture of different types of biosensors (e.g., a mixture of biosensorshaving polypeptide and polynucleotide selectivity components). Proteinmicroarrays are described, for example, in PCT Publication WO 00/04389,incorporated herein by reference. Examples of commercially availableprotein microarrays are those of Zyomyx of Hayward, Calif., CiphergenBiosystems, Inc. of Fremont, Calif. and Nanogen, Inc. of San Diego,Calif. Nucleic acid microarrays are described, for example, in U.S. Pat.Nos. 6,261,776 and 5,837,832. Examples of commercially available nucleicacid microarrays are those of Affymetrix, Inc. of Santa Clara, Calif.,BD Biosciences Clontech of Palo Alto, Calif. and Sigma-Aldrich Corp. ofSt. Louis, Mo.

3.E. Exemplary Embodiments Wherein the Selectivity Component isExpressed in the Cell or Tissue to be Analyzed

In other embodiments, the selectivity component is expressed within thecell or organism or subject to be analyzed. The expression methodsdescribed below may also be used to express a selectivity component in ahost cell that is then isolated and purified for use in the methods,wherein the biosensor is generated from a source external to the cell ortissue to be analyzed.

Generally, a nucleic acid encoding a selectivity component is introducedinto a host cell, such as by transfection or infection, and the hostcell is cultured under conditions allowing expression of the selectivitycomponent. Methods of introducing nucleic acids into prokaryotic andeukaryotic cells are well known in the art. Suitable media for mammalianand prokaryotic host cell culture are well known in the art. In someinstances, the nucleic acid encoding the subject polypeptide is underthe control of an inducible promoter, which is induced once the hostcells comprising the nucleic acid have divided a certain number oftimes. For example, where a nucleic acid is under the control of abeta-galactose operator and repressor, isopropylbeta-D-thiogalactopyranoside (IPTG) is added to the culture when thebacterial host cells have attained a density of about OD₆₀₀ 0.45-0.60.The culture is then grown for some more time to give the host cell thetime to synthesize the polypeptide. Cultures are then typically frozenand may be stored frozen for some time, prior to isolation andpurification of the polypeptide.

Thus, a nucleotide sequence encoding all or part of a selectivitycomponent may be used to produce a recombinant form of a selectivitycomponent via microbial or eukaryotic cellular processes. Ligating thesequence into a polynucleotide construct, such as an expression vector,and transforming, infecting, or transfecting into hosts, eithereukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterialcells), are standard procedures. Similar procedures, or modificationsthereof, may be employed to prepare recombinant polypeptides bymicrobial means or tissue-culture technology in accord with the subjectinvention.

Other embodiments of nucleic acid sequences encoding the selectivitycomponents, as well as vectors, host cells, and cultures thereof arefurther described below.

In another embodiment, the nucleic acid encoding a selectivity componentis operably linked to a bacterial promoter, e.g., the anaerobic E. coli,NirB promoter or the E. coli lipoprotein 11p promoter, described, e.g.,in Inouye et al. (1985) Nucl. Acids Res. 13:3101; Salmonella pagCpromoter (Miller et al., supra), Shigella ent promoter (Schmitt andPayne, J. Bacteriol. 173:816 (1991)), the tet promoter on Tn10 (Milleret al., supra), or the ctx promoter of Vibrio cholera. Any otherpromoter can be used in the invention. The bacterial promoter can be aconstitutive promoter or an inducible promoter. An exemplary induciblepromoter is a promoter which is inducible by iron or in iron-limitingconditions. In fact, some bacteria, e.g., intracellular organisms, arebelieved to encounter iron-limiting conditions in the host cytoplasm.Examples of iron-regulated promoters of FepA and TonB are known in theart and are described, e.g., in the following references: Headley, V. etal. (1997) Infection & Immunity 65:818; Ochsner, U. A. et al. (1995)Journal of Bacteriology 177:7194; Hunt, M. D. et al. (1994) Journal ofBacteriology 176:3944; Svinarich, D. M. and S. Palchaudhuri. (1992)Journal of Diarrhoeal Diseases Research 10:139; Prince, R. W. et al.(1991) Molecular Microbiology 5:2823; Goldberg, M. B. et al. (1990)Journal of Bacteriology 172:6863; de Lorenzo, V. et al. (1987) Journalof Bacteriology 169:2624; and Hantke, K. (1981) Molecular & GeneralGenetics 182:288.

In another embodiment, a signal peptide sequence is added to theconstruct, such that the selectivity component is secreted from cells.Such signal peptides are well known in the art.

In one embodiment, the powerful phage T5 promoter, that is recognized byE. coli RNA polymerase is used together with a lac operator repressionmodule to provide tightly regulated, high level expression orrecombinant proteins in E. coli. In this system, protein expression isblocked in the presence of high levels of lac repressor.

In one embodiment, the DNA is operably linked to a first promoter andthe bacterium further comprises a second DNA encoding a first polymerasewhich is capable of mediating transcription from the first promoter,wherein the DNA encoding the first polymerase is operably linked to asecond promoter. In a preferred embodiment, the second promoter is abacterial promoter, such as those delineated above. In an even morepreferred embodiment, the polymerase is a bacteriophage polymerase,e.g., SP6, T3, or T7 polymerase and the first promoter is abacteriophage promoter, e.g., an SP6, T3, or T7 promoter, respectively.Plasmids comprising bacteriophage promoters and plasmids encodingbacteriophage polymerases can be obtained commercially, e.g., fromPromega Corp. (Madison, Wis.) and InVitrogen (San Diego, Calif.), or canbe obtained directly from the bacteriophage using standard recombinantDNA techniques (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning:A Laboratory Manual, Cold Spring Laboratory Press, 1989). Bacteriophagepolymerases and promoters are further described, e.g., in the followingreferences: Sagawa, H. et al. (1996) Gene 168:37; Cheng, X. et al.(1994) Proc. Natl. Acad. Sci. USA 91:4034; Dubendorff, J. W. and F. W.Studier (1991) Journal of Molecular Biology 219:45; Bujarski, J. J. andP. Kaesberg (1987) Nucleic Acids Research 15:1337; and Studier, F. W. etal. (1990) Methods in Enzymology 185:60). Such plasmids can further bemodified according to the specific embodiment of the invention.

In another embodiment, the bacterium further comprises a DNA encoding asecond polymerase which is capable of mediating transcription from thesecond promoter, wherein the DNA encoding the second polymerase isoperably linked to a third promoter. In a preferred embodiment, thethird promoter is a bacterial promoter. However, more than two differentpolymerases and promoters could be introduced in a bacterium to obtainhigh levels of transcription. The use of one or more polymerase formediating transcription in the bacterium can provide a significantincrease in the amount of polypeptide in the bacterium relative to abacterium in which the DNA is directly under the control of a bacterialpromoter. The selection of the system to adopt will vary depending onthe specific use of the invention, e.g., on the amount of protein thatone desires to produce.

When using a prokaryotic host cell, the host cell may include a plasmidwhich expresses an internal T7 lysozyme, e.g., expressed from plasmid.Lysis of such host cells liberates the lysozyme which then degrades thebacterial membrane.

Other sequences that may be included in a vector for expression inbacterial or other prokaryotic cells include a synthetic ribosomalbinding site; strong transcriptional terminators, e.g., t0 from phagelambda and t4 from the rrnB operon in E. coli, to prevent read throughtranscription and ensure stability of the expressed polypeptide; anorigin of replication, e.g., ColE1; and beta-lactamase gene, conferringampicillin resistance.

Other host cells include prokaryotic host cells. Even more preferredhost cells are bacteria, e.g., E. coli. Other bacteria that can be usedinclude Shigella spp., Salmonella spp., Listeria spp., Rickettsia spp.,Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp.,Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp.,Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp.,Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp.,Helicobacter spp., Vibrio spp., Bacillus spp., and Erysipelothrix spp.Most of these bacteria can be obtained from the American Type CultureCollection (ATCC; 10801 University Blvd., Manassas, Va. 20110-2209).

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al., (1983)in Experimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83). These vectors may replicate in E. coli due the presenceof the pBR322 ori, and in S. cerevisiae due to the replicationdeterminant of the yeast 2 micron plasmid. In addition, drug resistancemarkers such as ampicillin may be used.

In certain embodiments, mammalian expression vectors contain bothprokaryotic sequences to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and17. In some instances, it may be desirable to express the recombinantprotein by the use of a baculovirus expression system. Examples of suchbaculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pFastBac-derived vectors.

In another variation, protein production may be achieved using in vitrotranslation systems. In vitro translation systems are, generally, atranslation system which is a cell-free extract comprising at least theminimum elements necessary for translation of an RNA molecule into aprotein. An in vitro translation system typically comprises at leastribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexesinvolved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex,comprising the cap-binding protein (CBP) and eukaryotic initiationfactor 4F (eIF4F). A variety of in vitro translation systems are wellknown in the art and include commercially available kits. Examples of invitro translation systems include eukaryotic lysates, such as rabbitreticulocyte lysates, rabbit oocyte lysates, human cell lysates, insectcell lysates and wheat germ extracts. Lysates are commercially availablefrom manufacturers such as Promega Corp., Madison, Wis.; Stratagene, LaJolla, Calif.; Amersham, Arlington Heights, Ill.; and GIBCO/BRL, GrandIsland, N.Y. In vitro translation systems typically comprisemacromolecules, such as enzymes, translation, initiation and elongationfactors, chemical reagents, and ribosomes. In addition, an in vitrotranscription system may be used. Such systems typically comprise atleast an RNA polymerase holoenzyme, ribonucleotides and any necessarytranscription initiation, elongation and termination factors. An RNAnucleotide for in vitro translation may be produced using methods knownin the art. In vitro transcription and translation may be coupled in aone-pot reaction to produce proteins from one or more isolated DNAs.

When expression of a carboxy terminal fragment of a polypeptide isdesired, i.e. a truncation mutant, it may be necessary to add a startcodon (ATG) to the oligonucleotide fragment comprising the desiredsequence to be expressed. It is well known in the art that a methionineat the N-terminal position may be enzymatically cleaved by the use ofthe enzyme methionine aminopeptidase (MAP). MAP has been cloned from E.coli (Ben-Bassat et al., (1987) J. Bacteriol. 169:751-757) andSalmonella typhimurium and its in vitro activity has been demonstratedon recombinant proteins (Miller et al., (1987) Proc. Natl. Acad. Sci.USA 84:2718-1722). Therefore, removal of an N-terminal methionine, ifdesired, may be achieved either in vivo by expressing such recombinantpolypeptides in a host which produces MAP (e.g., E. coli or CM89 or S.cerevisiae), or in vitro by use of purified MAP (e.g., procedure ofMiller et al.).

In cases where plant expression vectors are used, the expression of apolypeptide may be driven by any of a number of promoters. For example,viral promoters such as the 35S RNA and 19S RNA promoters of CaMV(Brisson et al., 1984, Nature, 310:511-514), or the coat proteinpromoter of TMV (Takamatsu et al., 1987, EMBO J., 6:307-311) may beused; alternatively, plant promoters such as the small subunit ofRUBISCO (Coruzzi et al., 1994, EMBO J., 3:1671-1680; Broglie et al.,1984, Science, 224:838-843); or heat shock promoters, e.g., soybean hsp17.5-E or hsp 17.3-B (Gurley et al., 1986, Mol. Cell. Biol., 6:559-565)may be used. These constructs can be introduced into plant cells usingTi plasmids, Ri plasmids, plant virus vectors; direct DNAtransformation; microinjection, electroporation, etc. For reviews ofsuch techniques see, for example, Weissbach & Weissbach, 1988, Methodsfor Plant Molecular Biology, Academic Press, New York, Section VIII, pp.421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed.,Blackie, London, Ch. 7-9.

An alternative expression system which can be used to express apolypeptide is an insect system. In one such system, Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes. The virus grows in Spodoptera frugiperda cells.The PGHS-2 sequence may be cloned into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of the coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed. (e.g., see Smith et al.,1983, J. Virol., 46:584, Smith, U.S. Pat. No. 4,215,051).

In a specific embodiment of an insect system, the DNA encoding thesubject polypeptide is cloned into the pBlueBacIII recombinant transfervector (Invitrogen, San Diego, Calif.) downstream of the polyhedrinpromoter and transfected into Sf9 insect cells (derived from Spodopterafrugiperda ovarian cells, available from Invitrogen, San Diego, Calif.)to generate recombinant virus. After plaque purification of therecombinant virus high-titer viral stocks are prepared that in turnwould be used to infect Sf9 or High Five™ (BTI-TN-5B1-4 cells derivedfrom Trichoplusia ni egg cell homogenates; available from Invitrogen,San Diego, Calif.) insect cells, to produce large quantities ofappropriately post-translationally modified subject polypeptide.Although it is possible that these cells themselves could be directlyuseful for drug assay, the subject polypeptides prepared by this methodcan be used for in vitro assays.

In another embodiment, the subject polypeptides are prepared intransgenic animals, such that in certain embodiments, the polypeptide issecreted, e.g., in the milk of a female animal.

Viral vectors may also be used for efficient in vitro introduction of anucleic acid into a cell. Infection of cells with a viral vector has theadvantage that a large proportion of the targeted cells can receive thenucleic acid. Additionally, polypeptides encoded by genetic material inthe viral vector, e.g., by a nucleic acid contained in the viral vector,are expressed efficiently in cells that have taken up viral vectornucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into mammals. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment of specialized cell lines (termed “packaging cells”) whichproduce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfer for gene therapy purposes(see Miller, A. D. (1990) Blood 76:271). Thus, recombinant retroviruscan be constructed in which part of the retroviral coding sequence (gag,pol, env) has been replaced by nucleic acid encoding one of theantisense E6AP constructs, rendering the retrovirus replicationdefective. The replication defective retrovirus is then packaged intovirions which can be used to infect a target cell through the use of ahelper virus by standard techniques. Protocols for producing recombinantretroviruses and for infecting cells in vitro or in vivo with suchviruses can be found in Current Protocols in Molecular Biology, Ausubel,F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections9.10-9.14, and other standard laboratory manuals. Examples of suitableretroviruses include pLJ, pZIP, pWE and pEM which are well known tothose skilled in the art. Examples of suitable packaging virus lines forpreparing both ecotropic and amphotropic retroviral systems includeCrip, Cre and Am. Retroviruses have been used to introduce a variety ofgenes into many different cell types, including neural cells, epithelialcells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bonemarrow cells, in vitro and/or in vivo (see for example Eglitis, et al.(1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl.Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci.USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

In choosing retroviral vectors as a gene delivery system for nucleicacids encoding the subject polypeptides, it is important to note that aprerequisite for the successful infection of target cells by mostretroviruses, and therefore of stable introduction of the geneticmaterial, is that the target cells must be dividing. In general, thisrequirement will not be a hindrance to use of retroviral vectors. Infact, such limitation on infection can be beneficial in circumstanceswherein the tissue (e.g., nontransformed cells) surrounding the targetcells does not undergo extensive cell division and is thereforerefractory to infection with retroviral vectors.

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example, PCT publications WO93/25234,WO94/06920, and WO94/11524). For instance, strategies for themodification of the infection spectrum of retroviral vectors include:coupling antibodies specific for cell surface antigens to the viral envprotein (Roux et al. (1989) Proc. Natl. Acad. Sci. USA 86:9079-9083;Julan et al. (1992) J. Gen Virol 73:3251-3255; and Goud et al. (1983)Virology 163:251-254); or coupling cell surface ligands to the viral envproteins (Neda et al. (1991) J Biol Chem 266:14143-14146). Coupling canbe in the form of the chemical cross-linking with a protein or othervariety (e.g., lactose to convert the env protein to anasialoglycoprotein), as well as by generating chimeric proteins (e.g.,single-chain antibody/env chimeric proteins). This technique, whileuseful to limit or otherwise direct the infection to certain tissuetypes, and can also be used to convert an ecotropic vector in to anamphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue- or cell-specific transcriptional regulatory sequenceswhich control expression of the genetic material of the retroviralvector.

Another viral gene delivery system utilizes adenovirus-derived vectors.The genome of an adenovirus can be manipulated such that it encodes agene product of interest, but is inactive in terms of its ability toreplicate in a normal lytic viral life cycle (see, for example, Berkneret al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155). Suitableadenoviral vectors derived from the adenovirus strain Ad type 5 dl324 orother strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are well knownto those skilled in the art. Recombinant adenoviruses can beadvantageous in certain circumstances in that they are capable ofinfecting non-dividing cells and can be used to infect a wide variety ofcell types, including airway epithelium (Rosenfeld et al. (1992) citedsupra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad.Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl.Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992)Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, the virusparticle is relatively stable and amenable to purification andconcentration, and, as above, can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in situations whereintroduced DNA becomes integrated into the host genome (e.g., retroviralDNA). Moreover, the carrying capacity of the adenoviral genome forforeign DNA is large (up to 8 kilobases) relative to other gene deliveryvectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol.57:267). Most replication-defective adenoviral vectors currently in useand therefore favored by the present invention are deleted for all orparts of the viral E1 and E3 genes but retain as much as 80% of theadenoviral genetic material (see, for example, Jones et al. (1979) Cell16:683; Berkner et al., supra; and Graham et al. in Methods in MolecularBiology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp.109-127). Expression of the inserted genetic material can be undercontrol of, for example, the E1A promoter, the major late promoter (MLP)and associated leader sequences, the E3 promoter, or exogenously addedpromoter sequences.

Yet another viral vector system useful for delivery of genetic materialencoding the subject polypeptides is the adeno-associated virus (AAV).Adeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle. (seeMuzyczka et al. Curr. Topics in Micro. and Immunol. (1992) 158:97-129).It is also one of the few viruses that may integrate its DNA intonon-dividing cells, and exhibits a high frequency of stable integration(see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol.7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlinet al. (1989) J. Virol. 62:1963-1973). Vectors comprising as little as300 base pairs of AAV can be packaged and can integrate. Space forexogenous DNA is limited to about 4.5 kb. An AAV vector such as thatdescribed in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can beused to introduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

In particular, a AAV delivery system suitable for targeting muscletissue has been developed by Gregorevic, et al., Nat Med. 2004 August;10(8):828-34. Epub 2004 July 25, which is able to ‘home-in’ on musclecells and does not trigger an immune system response. The deliverysystem also includes use of a growth factor, VEGF, which appears toincrease penetration into muscles of the gene therapy agent.

Other viral vector systems may be derived from herpes virus, vacciniavirus, and several RNA viruses.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of nucleicacids encoding the subject polypeptides, e.g. in a cell in vitro or inthe tissue of an animal. Most nonviral methods of gene transfer rely onnormal mechanisms used by mammalian cells for the uptake andintracellular transport of macromolecules. In preferred embodiments,non-viral gene delivery systems of the present invention rely onendocytic pathways for the uptake of genetic material by the targetedcell. Exemplary gene delivery systems of this type include liposomalderived systems, polylysine conjugates, and artificial viral envelopes.

In a representative embodiment, genetic material can be entrapped inliposomes bearing positive charges on their surface (e.g., lipofectins)and, optionally, which are tagged with antibodies against cell surfaceantigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka20:547-551; PCT publication WO91/06309; Japanese patent application1047381; and European patent publication EP-A-43075). For example,lipofection of papilloma-infected cells can be carried out usingliposomes tagged with monoclonal antibodies against PV-associatedantigen (see Viac et al. (1978) J Invest Dermatol 70:263-266; see alsoMizuno et al. (1992) Neurol. Med. Chir. 32:873-876).

In yet another illustrative embodiment, the gene delivery systemcomprises an antibody or cell surface ligand which is cross-linked witha gene binding agent such as polylysine (see, for example, PCTpublications WO93/04701, WO92/22635, WO92/20316, WO92/19749, andWO92/06180). For example, genetic material encoding the subject chimericpolypeptides can be used to transfect hepatocytic cells in vivo using asoluble polynucleotide carrier comprising an asialoglycoproteinconjugated to a polycation, e.g., polylysine (see U.S. Pat. No.5,166,320). It will also be appreciated that effective delivery of thesubject nucleic acid constructs via mediated endocytosis can be improvedusing agents which enhance escape of the gene from the endosomalstructures. For instance, whole adenovirus or fusogenic peptides of theinfluenza HA gene product can be used as part of the delivery system toinduce efficient disruption of DNA-comprising endosomes (Mulligan et al.(1993) Science 260-926; Wagner et al. (1992) Proc. Natl. Acad. Sci. USA89:7934; and Christiano et al. (1993) Proc. Natl. Acad. Sci. USA90:2122).

Tissue-specific expression of a selectivity component may be achieved byuse of a construct comprising a tissue-specific promoter.

4. Reporters

The reporter may be any molecule which produces a detectable signalchange in response to an alteration in the environment. For example; thesignal change may be an increase or decrease in signal intensity, or achange in the type of signal produced. In exemplary embodiments,suitable reporters include molecules which produce optically detectablesignals; including, for example, fluorescent and chemiluminescentmolecules. In certain embodiments, the reporter molecule is a longwavelength fluorescent molecule which permits detection of the reportersignal through a tissue sample, especially non-invasive detection of thereporter in conjunction with in vivo applications.

Without being bound by theory, in certain embodiments, the reportermolecule is a pH sensitive fluorescent dye (pH sensor dye) which shows aspectral change upon interaction of a selectivity component with atarget molecule. Interaction of the selectivity component with a targetmolecule may lead to a shift in the pH of the microenvironmentsurrounding the selectivity component due to the composition of acidicand basic residues on the selectivity and/or target molecules. In turn,the shift in the pH microenvironment leads to a detectable spectralchange in the signal of the pH sensitive fluorescent dye moleculeassociated with the selectivity component. In exemplary embodiments, apH sensitive dye is selected with an appropriate pKa to lead to anoptimal spectral change upon binding of the particular selectivitycomponent/target molecule combination. A variety of pH sensitive dyessuitable for use in accordance with the invention are commerciallyavailable. In exemplary embodiments, pH sensitive dyes include, forexample, fluorescein, umbelliferones (coumarin compounds), pyrenes,resorufin, hydroxy esters, aromatic acids, styryl dyes, tetramethylrhodamine dyes, and cyanine dyes, and pH sensitive derivatives thereof.

Without being bound by theory, in other embodiments, the reportermolecule is a polarity sensitive fluorescent dye (polarity sensor dye)which shows a spectral change upon interaction-of-a-selectivitycomponent with a target molecule. Interaction of the selectivitycomponent with a target molecule may lead to a shift in the polarity ofthe microenvironment surrounding the selectivity component due to thecomposition of polar and/or non-polar residues on the selectivity and/ortarget molecules. In turn, the change in the polarity of themicroenvironment leads to a detectable spectral change in the signal ofthe polarity sensitive fluorescent dye molecule associated with theselectivity component. A variety of polarity sensitive dyes suitable foruse in accordance with the invention are commercially available. Inexemplary embodiments, polarity sensitive dyes include, for example,merocyanine dyes,5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid(1,5-IAEDANS), and CPM, and polarity sensitive derivatives ofmerocyanine dyes, IAEDANS, and CPM.

Without being bound by theory, in certain embodiments, the reportermolecule is a fluorescent dye that is sensitive to changes in themicroviscosity of the local environment (restriction sensor dye).Interaction of the selectivity component with a target molecule may leadto a change in the microviscosity in the local environment surroundingthe selectivity component. In turn, the change in microviscosity maylead to a detectable spectral change in the signal of the mobilitysensor dye molecule associated with the selectivity component. Forexample, an increase of microviscosity upon target binding will restrictthe dye and increase the quantum yield of the emitted fluorescencesignal. A variety of restriction sensor dyes suitable for use inaccordance with the invention are commercially available. In exemplaryembodiments, restriction sensor dyes include, for example, monomethineand trimethine cyanine dyes, and microviscosity sensitive derivatives ofmonomethine and trimethine cyanine dyes.

Without being bound by theory, in certain embodiments, the reportermolecule is a fluorescent dye that exhibits a spectral change due to amodification in the tumbling rate of the dye as measured on a nanosecondtime scale (mobility sensor dye). Mobility sensor dye molecules may belinked to the selectivity component using a linker molecule that permitsfree rotation of the dye molecule. Upon interaction of the selectivitycomponent with a target molecule, the rotation of the dye moleculearound the linker may become restricted leading to a change in the ratioof parallel to perpendicular polarization of the dye molecule. A changein polarization of the mobility sensor dye may be detected as a changein the spectral emission of the dye and can be measured using lightpolarization optics for both excitation and emission to determine thetumbling rate of the dye. Abbott's fluorescence polarization technologyis an exemplary method for determining the polarization of the dye. Inexemplary embodiments, the mobility sensor dye is attached to theselectivity component using a triple-bond containing linker that extendsthe dye away from the surface of the selectivity component. A variety ofmobility sensor dyes suitable for use in accordance with the inventionare commercially available. In exemplary embodiments, mobility sensordyes include, for example, cyanine dyes and derivatives thereof.

In certain embodiments, the reporter molecule is a dye that exhibits achange in its spectral properties when specifically bound to aselectivity component. A nucleic acid, e.g. an aptamer, may be designedto specifically bind such a dye, for example Malachite Green (see R.Babendure, et al. (2003) J. Am. Chem. Soc. 125:14716). Such dyes, whenin complex with the nucleic acid or protein that is specific for them,change their spectral properties. For example, Malachite Green and itsanalogs, which is not normally fluorescent, becomes strongly fluorescentwhen bound to an aptamer specific for it or an scFv. FIG. 6 depicts thestructure of Malachite Green derivatized with a PEG amine.

In certain embodiments, the reporter molecule is represented bystructure I, II, or III:

wherein:

-   the curved lines represent the atoms necessary to complete a    structure selected from one ring, two fused rings, and three fused    rings, each said ring having five or six atoms, and each said ring    comprising carbon atoms and, optionally, no more than two atoms    selected from oxygen, nitrogen and sulfur;-   D, if present, is

-   m is 1, 2, 3 or 4, and for cyanine, oxynol and thiazole orange, m    can be 0;-   X and Y are independently selected from the group consisting of O,    S, and —C(CH₃)₂—;-   at least one R1, R2, R3, R4, R5, R6, or R7 is selected from the    group consisting of: a moiety that controls water solubility and    non-specific binding, a moiety that prevents the reporter molecule    from entering the cell through the membrane, a group that comprises,    optionally with a linker, biotin a hapten, a His-tag, or other    moiety to facilitate the process of isolating the selection entity,    a fluorescent label optionally comprising a linker, a photoreactive    group, or a reactive group such as a group containing    isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine,    mono- or di-halogen substituted pyridine, mono- or di-halogen    substituted diazine, phosphoramidite, maleimide, aziridine, sulfonyl    halide, acid halide, hydroxysuccinimide ester,    hydroxysulfosuccinimide ester, imido ester, hydrazine,    axidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide,    glyoxal, haloacetamido, or aldehyde;-   further-providing that R1 and R2 may be joined by a —CHR₈—CHR₈— or    —BF₂— biradical;    wherein;-   R₈ independently for each occurrence is selected from the group    consisting of hydrogen, amino, quaternary amino, aldehyde, aryl,    hydroxyl, phosphoryl, sulfhydryl, water solubilizing groups, alkyl    groups of twenty-six carbons or less, lipid solubilizing groups,    hydrocarbon solubilizing groups, groups promoting solubility in    polar solvents, groups promoting solubility in nonpolar solvents,    and -E-F; and further providing that any of R1, R2, R3, R4, R5, R6,    or R7 may be substituted with halo, nitro, cyan, —CO₂alkyl, —CO₂H,    —CO₂aryl, NO₂, or alkoxy,    wherein:-   F is selected from the group consisting of hydroxy, protected    hydroxy, alkoxy, sulfonate, sulfate, carboxylate, and lower alkyl    substituted amino or quartenary amino;-   E is spacer group of formula —(CH₂)n- wherein n is an integer from    0-5 inclusively;

In other embodiments, wherein m=0 in structures I, II, and III, thefollowing general structures IV, V and VI are afforded:

wherein:

-   the curved lines represent the atoms necessary to complete a    structure selected from one ring, two fused rings, and three fused    rings, each said ring having five or six atoms, and each said ring    comprising carbon atoms and, optionally, no more than two atoms    selected from oxygen, nitrogen and sulfur;-   D, if present, is

-   X and Y are independently selected from the group consisting of O,    S, and —C(CH₃)₂—;-   at least one R1, R2, R3, R4, R5, R6, or R7 is selected from the    group consisting of: a moiety that controls water solubility and    non-specific binding, a moiety that prevents the reporter molecule    from entering the cell through the membrane, a group that comprises,    optionally with a linker, biotin a hapten, a His-tag, or other    moiety to facilitate the process of isolating the selection entity,    a fluorescent label optionally comprising a linker, a photoreactive    group, or a reactive group such as a group containing    isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine,    mono- or di-halogen substituted pyridine, mono- or di-halogen    substituted diazine, phosphoramidite, maleimide, aziridine, sulfonyl    halide, acid halide, hydroxysuccinimide ester,    hydroxysulfosuccinimide ester, imido ester, hydrazine,    axidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide,    glyoxal, haloacetamido, or aldehyde;-   further-providing that R1 and R2 may be joined by a —CHR₈—CHR₈— or    —BF₂— biradical;    wherein;-   R₈ independently for each occurrence is selected from the group    consisting of hydrogen, amino, quaternary amino, aldehyde, aryl,    hydroxyl, phosphoryl, sulfhydryl, water solubilizing groups, alkyl    groups of twenty-six carbons or less, lipid solubilizing groups,    hydrocarbon solubilizing groups, groups promoting solubility in    polar solvents, groups promoting solubility in nonpolar solvents,    and -E-F; and further providing that any of R1, R2, R3, R4, R5, R6,    or R7 may be substituted with halo, nitro, cyan, —CO₂alkyl, —CO₂H,    —CO₂aryl, NO₂, or alkoxy wherein:-   F is selected from the group consisting of hydroxy, protected    hydroxy, alkoxy, sulfonate, sulfate, carboxylate, and lower alkyl    substituted amino or quartenary amino;-   E is spacer group of formula —(CH₂)n- wherein n is an integer from    0-5 inclusively;

The following are more specific examples of reporter molecules accordingto structure I, II, or III:

In these structures X and Y are selected from the group consisting of O,S and —CH(CH₃)₂—;

-   Z is selected from the group consisting of O and S;-   m is an integer selected from the group consisting of 0, 1, 2, 3 and    4 and, preferably an integer from 1-3.

In the above formulas, the number of methine groups determines in partthe excitation color. The cyclic azine structures can also determine inpart the excitation color. Often, higher values of m contribute toincreased luminescence and absorbance. At values of m above 4, thecompound becomes unstable. Thereupon, further luminescence can beimparted by modifications at the ring structures. When m=2, theexcitation wavelength is about 650 nm and the compound is veryfluorescent. Maximum emission wavelengths are generally 15-100 nmgreater than maximum excitation wavelengths.

The polymethine chain of the luminescent dyes of this invention may alsocontain one or more cyclic chemical groups that form bridges between twoor more of the carbon atoms of the polymethine chain. These bridgesmight serve to increase the chemical or photostability of the dye andmight be used to alter the absorption and emission wavelength of the dyeor change its extinction coefficient or quantum yield. Improvedsolubility properties may be obtained by this modification.

In certain embodiments, the reporter molecule is represented bystructure VII:

wherein:

-   W is N or C(R1);-   X is C(R2)₂;-   Y is C(R3)₂;-   Z is NR₁, O, or S;-   at least one R1, R2, or R3 is selected from the group consisting of:    a moiety that controls water solubility and non-specific binding, a    moiety that prevents the reporter molecule from entering the cell    through the membrane, a group that comprises, optionally with a    linker, biotin, a hapten, a His-tag, or other moiety to facilitate    the process of isolating the selection entity, a fluorescent label    optionally comprising a linker, a photoreactive group, or a reactive    group such as a group containing isothiocyanate, isocyanate,    monochlorotriazine, dichlorotriazine, mono- or di-halogen    substituted pyridine, mono- or di-halogen substituted diazine,    phosphoramidite, maleimide, aziridine, sulfonyl halide, acid halide,    hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido    ester, hydrazine, axidonitrophenyl, azide, 3-(2-pyridyl    dithio)-proprionamide, glyoxal, haloacetamido, or aldehyde;-   further providing that two R3 taken together may form O, S, NR1, or    N+(R1)₂; or two R3 along with R2 may form

-   wherein V is O, S, NR1, or N+(R1)₂; and-   further providing that any of R1, R2, or R3 may be substituted with    halo, nitro, cyano, —CO₂alkyl, —CO₂H, —CO₂aryl, NO₂, or alkoxy.

The following are more specific examples of reporter molecules accordingto structure VII:

In certain embodiments, the reporter molecule is represented bystructure VIII:

wherein:

-   at least one R1 is selected from the group consisting of: a moiety    that controls water solubility and non-specific binding, a moiety    that prevents the reporter molecule from entering the cell through    the membrane, a group that comprises, optionally with a linker,    biotin, a hapten, a His-tag, or other moiety to facilitate the    process of isolating the selection entity, a fluorescent label    optionally comprising a linker, a photoreactive group, or a reactive    group such as a group containing isothiocyanate, isocyanate,    monochlorotriazine, dichlorotriazine, mono- or di-halogen    substituted pyridine, mono- or di-halogen substituted diazine,    phosphoramidite, maleimide, aziridine, sulfonyl halide, acid halide,    hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido    ester, hydrazine, axidonitrophenyl, azide, 3-(2-pyridyl    dithio)-proprionamide, glyoxal, haloacetamido, or aldehyde;-   further providing that any two adjacent R1, in certain embodiments,    may be joined to form a fused aromatic ring; and-   further providing that R1, in certain embodiments, may be    substituted with halo, nitro, cyan, —CO₂alkyl, —CO₂H, —CO₂aryl, NO₂,    or alkoxy.

In certain embodiments, at least one, preferably only one, and possiblytwo or more of either R1, R2, R3, R4, R5, R6 and R7 groups in each ofthe foregoing moleculesis or contains a reactive group covalentlyreactive with amine, protected or unprotected hydroxy or sulfhydrylnucleophiles for attaching the dye to the labeled component. For certainreagents, at least one of said R1, R2, R3, R4, R5, R6 and R7 groups oneach molecule may also be a group that increases the solubility of thechromophore, or affects the selectivity of labeling of the labeledcomponent or affects the position of labeling of the labeled componentby the dye. Reactive groups that may be attached directly or indirectlyto the chromophore to form R1, R2, R3, R4, R5, R6 and R7 groups mayinclude reactive moieties such as groups containing isothiocyanate,isocyanate, monochlorotriazine, dichlorotriazine, -mono- or di-halogensubstituted pyridine, mono- or di-halogen substituted diazine,phosphoramidite, maleimide, aziridine, sulfonyl halide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester,hydrazine, axidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide,glyoxal, haloacetamido, and aldehyde.

Specific examples of R1, R2, R3, R4, R5, R6 and R7 groups that areespecially useful as reactive groups (e.g., for attaching dye to asubstrate, linkers, biotin, beads, microarrays, et alia) for labelingcomponents with available amino-, hydroxy-, and sulfhydryl groupsinclude:

wherein at least one of Q or W is a leaving group such as I, Br or Cland n is an integer from 0 to 4.

Specific examples of R1, R2, R3, R4, R5, R6 and R7 groups that areespecially useful for labeling components with available sulfhydrylswhich can be used for labeling selectivity components in a two-stepprocess are the following:

wherein Q is a leaving group such as I or Br, and wherein n is aninteger from 0 to 4.

Specific examples of R1, R2, R3, R4, R5, R6 and R7 groups that areespecially useful for labeling components by light-activated crosslinking include:

For the purpose of increasing water solubility or reducing unwantednonspecific binding of the labeled component to inappropriate componentsin the sample or to reduce the interactions between two or more reactivechromophores on the labeled component which might lead to quenching offluorescence, the R1, R2; R3, R4, R5, R6 and R7 groups can be selectedfrom the well known polar and electrically charged chemical groups.

In certain embodiments of the invention, the reporter molecule isrepresented by structure I, II, III, IV, V, VI, VII or VIII and theaccompanying definitions, and is a pH sensitive reporter molecule.

In certain embodiments of the invention, the reporter molecule isrepresented by structure I, II, III, IV, V, VI, VII or VIII and theaccompanying definitions, and is a polarity sensitive reporter molecule.

In certain embodiments of the invention, the reporter molecule isrepresented by structure I, II, III, IV, V, VI, VII or VIII and theaccompanying definitions, and is a microviscosity reporter molecule.

In certain embodiments of the invention, the reporter molecule isrepresented by structure I, II, III, IV, V, VI, VII or VIII and theaccompanying definitions, and is a mobility sensor reporter molecule.

In various other embodiments, the reporter is a of the type class IV:wherein the dye has the general structureA−B=A′

wherein A is selected from

wherein A′ is selected from

wherein R1, R2, R3, R4, R5 and R6 are selected from the group consistingof: a moiety that controls water solubility and non-specific binding, amoiety that prevents the dye from entering a cell through a membrane, agroup that comprises, optionally with a linker, biotin, a hapten, aHis-tag, or a moiety to facilitate isolation of the ligand, afluorescent label optionally comprising a linker, a photoreactive group,a reactive containing isothiocyanate, isocyanate, monochlorotriazine,dichlorotriazine, mono- or di-halogen substituted pyridine, mono- ordi-halogen substituted diazine, phosphoramidite, maleimide, aziridine,sulfonyl halide, acid halide, hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imido ester, hydrazine, axidonitrophenyl,azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal, haloacetamido, oraldehyde, and,

R1 and R2 may be joined by a —CHR₈—CHR₈— or —BF₂— biradical,

wherein R₈ independently for each occurrence is selected from the groupconsisting of hydrogen, amino, quaternary amino, aldehyde, aryl,hydroxyl, phosphoryl, sulfhydryl, water solubilizing groups, alkylgroups of twenty-six carbons or less, lipid solubilizing groups,hydrocarbon solubilizing groups, groups promoting solubility in polarsolvents, groups promoting solubility in nonpolar solvents, and -E-F,

wherein F is selected from the group consisting of hydroxy, protectedhydroxy, alkoxy, sulfonate, sulfate, carboxylate, and alkyl substitutedamino or quartenary amino and E is spacer group of formula —(CH₂)n-wherein n is an integer from 0-5 inclusively; and,

any of R1, R2, R3, R4, R5, R6, or R7 may be substituted with halo,nitro, cyan, —CO₂alkyl, —CO₂H, —CO₂aryl, NO₂, or alkoxy;

wherein B is

m is an integer from 0 to 4;

Z is selected from is H, C₁-C₁₈ alkyl, C₁-C₁₈ substituted alkyl, cyclicand heterocyclic having from one ring, two fused rings, and three fusedrings, each said ring having three to six atoms, and each said ringcomprising carbon atoms and from zero to two atoms selected from oxygen,nitrogen and sulfur and containing zero to 1 oxygen, nitrogen or sulfursubstituents attached; and,

Z′ is selected from H, C₁-C₁₈ alkyl, C₁-C₁₈ substituted alkyl, cyclicand heterocyclic having from one ring, two fused rings, and three fusedrings, each said ring having three to six atoms, and each said ringcomprising carbon atoms and from zero to two atoms selected from oxygen,nitrogen and sulfur, A, and A′. (See FIG. 2.)

In various embodiments, the spectral change of the sensor dye uponinteraction of the selectivity component and a target molecule mayinclude, for example, a shift in absorption wavelength, a shift inemission wavelength, a change in quantum yield, a change in polarizationof the dye molecule, and/or a change in fluorescence intensity. Thechange can be two-fold, ten-fold, one hundred-fold, one thousand-fold oreven higher. Any method suitable for detecting the spectral changeassociated with a given sensor dye may be used in accordance with theinventions. In exemplary embodiments, suitable instruments for detectionof a sensor dye spectral change, include, for example, fluorescentspectrometers, filter fluorometers, microarray readers, optical fibersensor readers, epifluorescence microscopes, confocal laser scanningmicroscopes, two photon excitation microscopes, and flow cytometers.

In variant embodiments, the reporter molecule may be associated with theselectivity component or the target molecule. In exemplary embodiments,the reporter molecule is covalently attached to the selectivitycomponent. The reporter molecule may be covalently attached to theselectivity component using standard techniques. In certain embodimentsthe reporter molecule may be directly attached to the selectivitycomponent by forming a chemical bond between one or more reactive groupson the two molecules. In an exemplary embodiment, a thiol reactivereporter molecule is attached to a cysteine residue (or other thiolcontaining molecule) on the selectivity component. Alternatively, thereporter molecule may be attached to the selectivity component via anamino group on the selectivity component molecule. In other embodiments,the reporter molecule may be attached to the selectivity component via alinker group. Suitable linkers that may be used in accordance with theinventions include, for example, chemical groups, an amino acid or chainof two or more amino acids, a nucleotide or chain of two or morepolynucleotides, polymer chains, and polysaccharides. In exemplaryembodiments, the reporter molecule is attached to the selectivitycomponent using a linker having a maleimide moiety. Linkers may behomofunctional (containing reactive groups of the same type),heterofunctional (containing different reactive groups), orphotoreactive (containing groups that become reactive on illumination).A variety of photoreactive groups are known, for example, groups in thenitrene family.

In various embodiments, one or more reporter molecules may be attachedat one or more locations on the selectivity component. For example, twoor more molecules of the same reporter may be attached at differentlocations on a single selectivity component molecule. Alternatively, twoor more different reporter molecules may be attached at differentlocations on a single selectivity component molecule. In exemplaryembodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more reporter molecules areattached at different sites on the selectivity component. The one ormore reporter molecules may be attached to the selectivity component soas to maintain the activity of the reporter molecule and the selectivitycomponent.

In certain embodiments, the location of the reporter molecule isoptimized to permit exposure of the reporter molecule to changes in themicroenvironment upon interaction of the selectivity component with atarget molecule while maintaining the ability of the selectivitycomponent to interact with the target molecule. In exemplaryembodiments, the reporter molecule is attached to the selectivitycomponent in spatial proximity to the target binding site withoutaffecting the ability of the selectivity component to interact with thetarget molecule.

In certain embodiments, the reporter molecule further comprises a moietythat is specific for the selectivity component. For example, thereporter molecule may be linked to a substrate, a hapten, etc. that isspecific for the selectivity component if it is an enzyme,hapten-binding protein, etc. The reporter molecule may be covalentlyattached to the moiety using standard techniques. In certain embodimentsthe reporter molecule may be directly attached to the moiety by forminga chemical bond between one or more reactive groups on the twomolecules. In other embodiments, the reporter molecule may be attachedto the moiety via a linker group. Suitable linkers that may be used inaccordance with the inventions include, for example, chemical groups, anamino acid or chain of two or more amino acids, a nucleotide or chain oftwo or more polynucleotides, polymer chains, and polysaccharides.Linkers may be homofunctional (containing reactive groups of the sametype), heterofunctional (containing different reactive groups), orphotoreactive (containing groups that become reactive on illumination).

5. Exemplary Uses

The biosensors of the invention may be used to detect and/or quantitateanalytes in any solid, liquid or gas sample, as well as in any cell ortissue or organism of interest.

In various exemplary embodiments, the biosensors of the invention may beused for a variety of diagnostic and/or research applications,including, for example, monitoring the development of engineeredtissues, in vivo monitoring of analytes of interest (includingpolynucleotides, polypeptides, hormones, lipids, carbohydrates, andsmall inorganic and organic compounds and drugs) using injectable freebiosensors or implants functionalized with one or more biosensors,biological research (including developmental biology, cell biology,neurobiology, immunology, and physiology); detection of microbial, viraland botanical polynucleotides or polypeptides, drug discovery, medicaldiagnostic testing, environmental detection (including detection ofhazardous substances/hazardous wastes, environmental pollutants,chemical and biological warfare agents, detection of agriculturaldiseases, pests and pesticides and space exploration), monitoring offood freshness and/or contamination, food additives, and food productionand processing streams, monitoring chemical and biological products andcontaminants, and monitoring industrial and chemical production andprocessing streams.

In one embodiment, the biosensors described herein may be used fordetecting environmental pollutants, including, air, water and soilpollutants. Examples of air pollutants, include, for example, combustioncontaminants such as carbon monoxide, carbon dioxide, nitrogen dioxide,sulfur dioxide, and tobacco smoke; biological contaminants such asanimal dander, molds, mildew, viruses, pollen, dust mites, and bacteria;volatile organic compounds such as formaldehyde, fragrance products,pesticides, solvents, and cleaning agents; heavy metals such as lead ormercury; and asbestos, aerosols, ozone, radon, lead, nitrogen oxides,particulate matter, refrigerants, sulfur oxides, and volatile organiccompounds. Examples of soil pollutants, include, for example, acetone,arsenic, barium, benzene, cadmium, chloroform, cyanide, lead, mercury,polychlorinated biphenyls (PCBs), tetrachloroethylene, toluene, andtrichloroethylene (TCE). Examples—of water pollutants, include, forexample, arsenic, contaminated sediment, disinfection byproducts,dredged material, and microbial pathogens (e.g., Aeromonas, Coliphage,Cryptosporidium, E. coli, Enterococci, Giardia, total coliforms,viruses).

In other embodiments, the biosensors described herein may be used fordetecting hazardous substances, including, for example, arsenic, lead,mercury, vinyl chloride, polychlorinated biphenyls (PCBs), benzene,cadmium, benzopyrene, polycyclic aromatic hydrocarbons,benzofluoranthene, chloroform, DDT, aroclors, trichloroethylene,dibenz[a,h]anthracene, dieldrin, hexavalent chromium, chlordane,hexachlorobutadiene, etc.

In another embodiment, the biosensors described herein may be used fordetecting chemical and biological warfare agents. Examples of biologicalwarfare agents, include, for example, bacteria such as anthrax (Bacillusanthracis), botulism (Clostridium botulinum toxin), plague (Yersiniapestis), tularemia (Francisella tullarensis), brucellosis (Brucellaspecies), epsilon toxin from Clostridium peringens, food safety threats(e.g., Salmonella species, Escherichia coli 0157:H7, Shigella); watersafety threats (e.g., Vibrio cholerae and Cryptosporidium parvum),glanders (Burkholderia mallei), Melioidosis (Burkholderia pseudomallei),Psittacosis (Chlamydia psittaci), Q fever (Coxiella burnetii), Ricintoxin from Ricinus communis, Staphylococcal enterotoxin B, Typhus fever(Rickettsia prowaaekii) and viruses, such as filoviruses (e.g., ebola orMarburg), arenaviruses (e.g., Lassa and Machupo), hantavirus, smallpox(variola major), hemorrhagic fever virus, Nipah virus, and alphaviruses(e.g., Venezuelan equine encephalitis, eastern equine encephalitis,western equine encephalitis). Examples of chemical warfare agents,include for example, blister agents (e.g., distilled mustard, lewisite,mustard gas, nitrogen mustard, phosgene oxime, ethyldichloroarsine,methyldichloroarsine, phenodichloroarsine, sesqui mustard), bloodpoisoning agents (arsine, cyanogen chloride, hydrogen chloride, hydrogencyanide), lung damaging agents (chlorine, diphosgene, cyanide, nitrogenoxide, perfluorisobutylene, phosgene, red phosphorous, sulfurtrioxide-chlorosulfonic acid, teflon, titanium tetrachloride, zincoxide), incapacitating agents (agent 15, BZ, canniboids, fentanyls, LSD,phenothiazines), nerve agents (cyclohexyl sarin, GE, Soman, Sarin,Tabun, VE, VG, V-Gas, VM, VX), riot control/tear gas agents(bromobenzylcyanide, chloroacetophenone, chloropicrin, CNB, CNC, CNS,CR, CS), and vomit inducing agents (adamsite, diphenylchloroarsine,diphenylcyanoarsine).

In another embodiment, the biosensors described herein may be used formonitoring food freshness and/or contamination, food additives, and foodproduction and processing streams: Examples of bacterial contaminantsthat may lead to foodborne illnesses include, for example, Bacillusanthracis, Bacillus cereus, Brucella abortus, Brucella melitensis,Brucella suis, Campylobacter jejuni, Clostridium botulinum, Clostridiumperfringens, enterohemorrhagic E. coli (including E. coli0157:H7-and-other Shiga toxin-producing; E. coli), enterotoxigenic E.coli, Listeria monocytogenes, Salmonella, Shigella, Staphylococcusaureus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus,Yersinia enterocolytica and Yersinia pseudotuberculosis. Examples ofviral contaminants that may lead to foodborne illnesses include, forexample, hepatitis A, norwalk-like viruses, rotavirus, astroviruses,calciviruses, adenoviruses, and parvoviruses. Examples of parasiticcontaminants that may lead to foodborne illnesses include, for example,Cryptosporidium parvum, Cyclospora cayetanensis, Entamoeba histolytica,Giardia lamblia, Toxoplasma gondii, and Trichinella spiralis. Examplesof noninfectious toxins or contaminants that may lead to foodborneillnesses include, for example, antimony, arsenic, cadmium, ciguateratoxin, copper, mercury, museinol, muscarine, psilocybin, copriusartemetaris, ibotenic acid, amanita, nitrites, pesticides(organophosphates or carbamates), tetrodotoxin, scombroid, shellfishtoxins, sodium floride, thallium, tin, vomitoxin, and zinc.

In one embodiment, the biosensors described herein may be used for invitro and/or in vivo monitoring of analytes of interest. The biosensorsmay be injected or otherwise administered to a patient as free moleculesor may be immobilized onto a surface before introduction into a patient.When administered. as free molecules, the biosensors may be used todetect analytes of interest in both interstitial spaces and insidecells. For detection of analytes inside of cells, the selectivitycomponent may be modified, as described above, with a tag thatfacilitates translocation across cellular membranes. Alternatively, theselectivity components may be introduced into cells using liposomedelivery methods or mechanical techniques such as direct injection orballistic-based particle delivery systems (see for example, U.S. Pat.No. 6,110,490). In other embodiments, the biosensors may be immobilizedonto a surface (including, for example, a bead, chip, plate, slide,strip, sheet, film, block, plug, medical device, surgical instrument,diagnostic instrument, drug delivery device, prosthetic implant orother-structure) and then introduced into a patient, for example, bysurgical implantation. In exemplary embodiments, the biosensors areimmobilized onto the surface of an implant, such as an artificial orreplacement organ, joint, bone, or other implant. The biosensors of theinvention may also be immobilized onto particles, optical fibers, andpolymer scaffolds used for tissue engineering. For example, one or morebiosensors may be immobilized onto a fiber optic probe for precisepositioning in a tissue. The fiber optic then provides the pathway forexcitation light to the sensor tip and the fluorescence signal back tothe photodetection system. In still other embodiments, at least theselectivity component of the biosensor is transfected in cell or otherhost organism of interest and expressed within the cell or other hostorganism of interest.

In each of the various embodiments of the invention, a single biosensormay be used for detection of a single target molecule or two or morebiosensors may be used simultaneously for detection of two or moretarget molecules. For example, 2, 5, 10, 20, 50, 100, 1000, or moredifferent selectivity components may be used simultaneously fordetection of multiple targets.

When using multiple selectivity components simultaneously, eachselectivity component may be attached to a different reporter moleculeto permit individual detection of target binding to each selectivitycomponent. Alternatively, a dual detection system may be used where twoor more selectivity components may be attached to the same reportermolecule (for example, the same sensor dye) and be identified based on asecond detectable signal. For example, selectivity components havingdifferent target specificities but containing the same sensor dye may bedistinguished based on the signal from a color coded particle to whichit is attached. The readout for each selectivity component involvesdetection of the signal from the sensor dye, indicating association withthe target molecule, and detection of the signal from the color codedparticle, permitting identification of the selectivity component. In anexemplary embodiment, a panel of biosensors may be attached to a varietyof color coded particles to form a suspension array (LuminexCorporation, Austin, Tex.). The mixture of coded particles associatedwith the biosensors of the invention may be mixed with a biologicalsample or administered to a patient. Flow cytometry or microdialysis maythen be used to measure the signal from the sensor dye and to detect thecolor code for each particle. In various embodiments, the identificationsignal may be from a color coded particle or a second reporter molecule,including, for example, chemiluminescent, fluorescent, or other opticalmolecules, affinity tags, and radioactive molecules.

In other embodiments, one or more biosensors of the invention may beimmobilized onto a three dimensional surface suitable for implantationinto a patient. The implant allows monitoring of one or more analytes ofinterest in a three dimensional space, such as, for example, the spacesbetween tissues in a patient.

In other embodiments, the biosensors of the invention may be exposed toa test sample. Any test sample suspected of containing the target may beused, including, but not limited to, tissue samples such as biopsysamples and biological fluids such as blood, sputum, urine and semensamples, bacterial cultures, soil samples, food samples, cell cultures,etc. The target may be of any origin, including animal, plant ormicrobiological (e.g., viral, prokaryotic, and eukaryotic organisms,including bacterial, protozoal, and fungal, etc.) depending on theparticular purpose of the test. Examples include surgical specimens,specimens used for medical diagnostics, specimens used for genetictesting, environmental specimens, cell culture specimens, foodspecimens, dental specimens and veterinary specimens. The sample may beprocessed or purified prior to exposure to the biosensor(s) inaccordance with techniques known or apparent to those skilled in theart.

In other embodiments, the biosensors of the invention may be used todetect bacteria and eucarya in food, beverages, water, pharmaceuticalproducts, personal care products, dairy products or environmentalsamples. The biosensors of the invention are also useful for theanalysis of raw materials, equipment, products or processes used tomanufacture or store food, beverages, water, pharmaceutical products,personal care products, dairy products or environmental samples.

Alternatively, the biosensors of the invention may be used to diagnose acondition of medical interest. For example the methods, kits andcompositions of this invention will be particularly useful for theanalysis of clinical specimens or equipment, fixtures or products usedto treat humans or animals. In one preferred embodiment, the assay maybe used to detect a target sequence which is specific for a geneticallybased disease or is specific for a predisposition to a genetically baseddisease. Non-limiting examples of diseases include, beta-thalassemia,sickle cell anemia, Factor-V Leiden, cystic fibrosis and cancer relatedtargets such as p53, p 10, BRC-1 and BRC-2. In still another embodiment,the target sequence may be related to a chromosomal DNA, wherein thedetection, identification or quantitation of the target sequence can beused in relation to forensic techniques such as prenatal screening,paternity testing, identity confirmation or crime investigation.

In still other embodiments, the methods of the invention include theanalysis or manipulation of plants and genetic materials derivedtherefrom as well as bio-warfare reagents. Biosensors of the inventionwill also be useful in diagnostic applications, in screening compoundsfor leads which might exhibit therapeutic utility (e.g. drugdevelopment) or in screening samples for factors useful in monitoringpatients for susceptibility to adverse drug interactions (e.g.pharmacogenomics).

In certain embodiments, the biosensors of the invention, or nucleicacids encoding them, may be formulated into a pharmaceutical compositioncomprising one or more biosensors and a pharmaceutically acceptablecarrier, adjuvant, or vehicle. The term “pharmaceutically acceptablecarrier” refers to a carrier(s) that is “acceptable” in the sense ofbeing compatible with the other ingredients of a composition and notdeleterious to the recipient thereof. Methods of making and using suchpharmaceutical compositions are also included in the invention. Thepharmaceutical compositions of the invention can be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally, or via an implanted reservoir. The term“parenteral” as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intrasynovial, intrasternal,intrathecal, intralesional, and intracranial injection or infusiontechniques.

In other embodiments, the invention contemplates kits including one ormore biosensors of the invention, and other subject materials, andoptionally instructions for their use. Uses for such kits include, forexample, environmental and/or biological monitoring or diagnosticapplications.

A biosensor, or an isolated, purified biosensor, comprising theselectivity component may have at least about 85% sequence identity withSEQ ID NO:1 through 72. The selectivity component may reversibly bindeither a monomethin cyanine dye, or Malachite Green, or an analogthereof A host cell may express the biosensor. Further, a vector may becomprised of a nucleic acid sequence having at least about 85%, andpreferably 95%, sequence identity to a polynucleotide encoding a proteinwith SEQ ID NO:1 through 72. A host cell may comprise the vector.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.Provided are several single chain antibody (scFv) based sensors thatcomprise amino acid sequences that lead to specific binding of certainmonomethin cyanine dyes (TO1 and its analogs (the structure of TO1derivatized with PEG amine is shown in FIG. 7) and Malachite Green (andits analogs) in a way that produces a large increase in fluorescence ofthe dye (a “fluorogen”) when it is in the bound state.

Example 1 scFv-Based Sensors that Reversibly Bind Fluorescent Dyes

In one experiment, an scFv that reversibly binds noncovalently the dyeTO1 (“scFv1”) was produced. See, for example, SEQ ID NO:1. A 2700-foldincrease in fluorescence was detected (by methods described below) whenthe TO1 binds to the scFv. This sequence was used in subsequent work toproduce additional scFvs that bind to TO1 with a stronger bindingaffinity and have enhanced fluorescence. In other experiments, scFvswere developed that bind certain derivatives of malachite green wherethe noncovalent binding is strong and again a large increase influorescence is produced upon binding of the dye to the scFv. See, FIGS.9, 10A and 10B.

1A

The genetic sequence of this scFv may be inserted into genes for otherproteins so that the expressed fusion protein will contain thedye-binding scFv. For example, a cell surface membrane protein waslabeled with scFv1 by genetic methods and expressed on the cell surfaceof cultured mammalian cells. When the relatively non-fluorescent (inwater or buffer) dye TO1 was added to cell suspensions containing theprotein-scFv, a fraction of the dye binds to the scFv with a large (morethan 2500×) fluorescence increase upon binding and thereby produced afluorescent label on the protein of interest on the membrane surface.Thus, the appearance of fluorescence when the scFv and the dye are bothpresent is very rapid. Thus, TO1 and the corresponding TO1 scFv may beused to detect protein expression of the scFv-fusion protein on thesurface of the cell where the scFv will have access to the membraneimpermeant dye that has been added to the culture medium. Other proteinsmay be labeled with the scFv and if the labeled protein is presentinside the cell, a membrane permeant fluorogen may be used to detect thepresence of that particular scFv fusion protein.

The presence of the scFv can be used to ascertain the amount,expression, degradation, or location of the fusion protein.

In one experiment, yeast FAPs were fused via AGA2 to the AGA1-AGA2complex, which is directed to the outer leaflet of the plasma membraneby a C-terminal glycosylphosphatidylinositol (GPI) anchor beforeinsertion into the cell wall. Live cell imaging using fluorescentproteins fused to GPIs or GPI-anchored proteins is useful for studyingorganization and function of membrane proteins, including signalingreceptors and cell adhesion molecules, but these constructs may alsolabel cell structures involved in biosynthesis, secretion anddegradation. Dynamic imaging of lipid rafts and other surface featureswould benefit by confining fluorescence to proteins anchored to theouter leaflet. Whereas methods such as total internal reflectionfluorescence microscopy have evolved to allow selective observation,there are no methods for selective labeling and homogenous detection. Toillustrate such labeling and detection, an MG FAP and an identicallyanchored AGA2-GFP fusion protein was imaged. The MG FAP and the GFP werevisualized on the extracellular surface, but intracellular structures,many with the morphology expected of vacuoles and nuclear membranes(endoplasmic reticulum), were visualized only by the GFP. To explorefluorogenic labeling of mammalian cell surface proteins, selected TO1and MG FAPs were fused to the N terminus of platelet-derived growthfactor receptor (PDGFR) transmembrane domain. The TO1 and MG FAPs werethan expressed stably in NIH3T3 mouse fibroblasts and in M21 human renalcarcinoma cells. In each case the transfected cells exhibited strongsurface fluorescence when exposed to low concentrations of TO1-2p orMG-11p fluorogen. No significant intracellular fluorescence was observedunder these experimental conditions; TO1-2p and MG-11p controls did notenter living NIH3T3 cells. It is noteworthy that little or nophotobleaching of cell surface fluorescence was observed. Separateexperiments showed that TO1-2p FAPs resisted bleaching about as well asEGFP and that MG FAPs were even more bleach resistant (FIG. 16).Fluorescence signal of TO1-2p FAPs decayed to a TO1-2p concentrationdependent steady state, which suggests without being bound by theory,that rapid exchange of fluorogen (and/or fluorogen photoproducts)between the solution and the FAP itself was effectively buffering thesystem against photobleaching. For MG FAPs, other mechanisms maycontribute, such as loose sequestration of dark fluorogen on the plasmamembrane outer surface. In aqueous solution mobile fluorogens such as MGor TO1 show almost no photoreactivity, but under illumination MGconjugated to an antibody generates reactive oxygen species at a ratesimilar to GFP, sufficient to be phototoxic under continuous or intenseexcitation. Phototoxicity correlates with photobleaching, suggestingthat MG and TO1 FAPs generated reactive oxygen species at GFP-likerates. MG has also been used as an antifungal agent; at experimentalconcentrations the MG derivatives studied here had little or no effecton yeast growth (FIG. 17). Cell surface-exposed FAPs visualized with amembrane-impermeant fluorogen were seen at the plasma membrane only.When exposed to a membrane-permeant fluorogen, however, these same cellsshowed additional fluorescence within elements of the secretoryapparatus, including the nuclear endoplasmic reticulum and the Golgi.This result suggests that permeant fluorogens can be used to visualizeFAPs shortly after cotranslational insertion into the lumen, and thuspotentially report protein folding in near real time. Permeantfluorogens can be added and withdrawn at will, facilitating developmentof pulse-chase and other approaches to studying secretory and endocyticpathways. When incorporated into fusion proteins, FAP domains provided areporter of protein location and abundance in time and space.Fluorescence signal was generated only upon addition of a secondcomponent (the fluorogen); in this respect FAPs resemble thesite-specific chemical labeling systems. However, all chemical labelingsystems require additional manipulation such as enzymatic conjugationsteps or washes to reduce background signal, whereas FAPs can bevisualized directly after fluorogen addition on a time scale of seconds(on the cell surface) to minutes (within the secretory apparatus).Fluorescence visualization can also be spatially controlled by theappropriate choice of fluorogen, enabling one to selectively observefusion proteins at particular cellular locations. Multicolor imaging ofspectrally and antigenically distinct FAPs co-expressed within a cellwill greatly enhance the usefulness of different fluorogens todynamically monitor complex cellular functions. ScFv-based FAPs containinternal disulfide linkages and are currently adapted for use only innonreducing environments, mainly the cell surface and secretoryapparatus. FAP/fluorogens thus complement the biarsenical system, whichis generally limited to intracellular reducing environments, primarilythe cytoplasmic and nuclear compartments. However, it has been shownthat functional scFvs can be expressed cytoplasmically in adisulfide-free format in yeast and mammalian cells, and futuredevelopments of scFv and other FAP platforms will address theseintracellular compartments.

1B

Additionally, the genetic sequence of an scFv may be used to createmolecular biosensors. In one example, the initial sequence of an scFv ismodified to make the binding of a reporter molecule by the scFvsensitive to the presence of other molecular interactions with the scFv.Such interactions include contact with another protein or peptide. Suchinteractions can involve contact with a kinase or phosphatase or othercovalent modification that alters an amino acid of the scFv to produce achange in fluorogen fluorescence. The interaction can produce a stericchange or allosteric change near the reporter group on the sensor thatproduces the fluorescence signal. The interaction can alter a chargedamino acid side chain or an ionizable group on a side chain or hydrogenbonding group or a non-polar group near the reporter that produces afluorescence signal.

1C

The TO1 scFv was created using a large PNNL Yeast Display librarycomprised of cells that each express on its surface on of an estimated10⁹ different sequence variations in the heavy and light variableregions that constitute the displayed scFv proteins. The TO1-bindingscFv was isolated in two steps. First, a TO1 dye linked via a PEGpolymer to a biotin was used in a magnetic bead separation procedure toisolate a population of cells that was enriched for cells that bind invariable degrees to the TO1 dye. In a second step, flow cytometryexperiments were carried out to select for cells that bind the dye andmake it highly fluorescent. One of the highly fluorescent cells wassorted and cloned. This yeast cell was the source of the scFv. Yeastplasmid DNA encoding this scFv clone was amplified by PCR methods, andthen sequenced. Flow cytometry data has demonstrated the successfulcloning of this highly fluorescent TO1-binding scFv. Similarly formalachite green.

1D

It is possible to engineer and select other proteins that are capable ofbinding to the fluorescent reporters of this invention such that theyalso would function as biosensors in a way similar to the scFv1 of thisexample. Such engineered proteins that can be used as biosensors we callFluorescent Binding Protein (FBP) sensors. Examples of suchnon-immunological binding proteins engineered to bind small-to-mediummolecular weight molecules are described by Bintz et al. (2005) NatureBiotech. 23:1257-1268.

Example 2 Reporter Molecule-Localizing System Based on SAb scFvTechnology

In this example, a combination of reagents with which the location ofthe reporter molecule can be controlled within a cell by genetic methods

In biological research and pharmaceutical drug discovery it is useful tobe able to target specific reporter molecules to certain regions orstructures within a cell. Examples of such specific reporter moleculesinclude regulatory metabolites such as small peptides, growth factors,and inhibitory RNAs. Also such reporter molecules s may includesynthetic and natural molecules that modify cellular behavior such asthose often used and developed by the pharmaceutical industry. Further,such reporter molecules might also include fluorescent or lightabsorbing molecules that provide a signal for targeting of a proteinwithin a cell or that are sensitive to a physiological property of thecell such as membrane potential, ion concentration or enzyme activity.In other words, the reporter molecules are used to modulate or perturbthe activity of specific proteins, pathways and networks within cells orto measure biochemical, structural and physiological properties ofcells. The reporter molecules allow control of targeting of the regionof a cell or tissue where the perturbation or measurement occurs.

Many types of reporter molecules diffuse into cells and perturb orreport from many regions of a cell where the probe non-specificallyassociates. The targeting in the reporter molecules may be controlled bybringing together two molecular entities within a cell or on its surfaceor in a tissue. One such entity may comprise the reporter molecule, ahapten and a water soluble linker that separates the probe and thehapten. The hapten is a molecule with which antibodies that bind themolecule can be generated by known procedures. This three part structureis shown on the right hand side of FIG. 3. Such a reporter might be avoltage sensitive dye, a calcium indicator, a pH indicator, ionindicator, polarity indicator, mechanical stress indicator or anindicator of some other physiological or molecular process occurring inthe cell.

The second entity required for targeting the probe in a cell consists ofa selectivity component, e.g., a hapten-binding antibody, specifically ascFv that may be genetically fused to a “protein of interest” within thecell. The protein of interest serves the purpose of localizing thereporter molecule-binding scFv, and thus the reporter molecule after ithas been incorporated in the cell or tissue, to a specific region ofinterest within or on a cell. The protein of interest and the scFv areillustrated on the left hand side of FIG. 3.

The process of targeting the reporter molecule within the cell takesplace in several steps: (1) first, a hapten-linker-reporter moleculemust be synthesized, then (2) a hapten-binding scFv must be created.This may be done, for example, by yeast selection procedures developedby Wittrup, et al. (Methods Enzymol. (2000) 328:430-33; Proc. Natl.Acad. Sci. USA (2000) 97:10701-5; Nucleic Acids Res (2004) 32:e36). (3)The cell of interest must be transfected with a gene that codes for theprotein of interest fused to the hapten-binding protein. (4) Thehapten-linker-reporter molecule must be incorporated into the cell bydiffusion through the membrane or microinjection or by another means.

The result of this process will be noncovalent placement of the reportermolecule into the protein of interest that may be in the nucleus, or anorganelle, in the cytoplasm, on the internal surface of the membrane, orin a specific location of a tissue. By using a photoreactive group or areactive functional group the reporter moiety may be covalently attachedto the protein of interest.

Several variations are possible:

Variation 1: Non-Covalent Binding of the Hapten-Linker-Reporter Moleculeto the scFv.

The affinity of this binding depends on the structure of the scFv andthe hapten. In some cases, it is desirable to have relatively weakbinding so that the reporter molecule can experience the targetedprotein of interest but also other regions of the cell. In other cases,it is desirable to have strong binding so that the probe ispredominantly at the scFv. In this case, there would be lessnon-specific binding of the hapten-linker-reporter molecule to otherregions of the tissue and the modulating or measuring capabilities ofthe reporter molecule would be targeted mainly to the scFv-protein ofinterest region. There would therefore be a better signal-to-noise inthe experiment. The binding constant of the hapten to the antibody(often expressed as a dissociation constant, Kd) can be adjusted by theprocedures used to create the hapten-binding scFv. By yeast selectionprocedures and by a process called affinity maturation (where the scFvis genetically mutated and further selection is carried out) it ispossible to considerably decrease Kd (increase the binding affinity).

Variation 2: Covalent Linkage of the Hapten-Linker-Reporter Molecule tothe scFv Using Light.

In this case, the hapten is modified to contain a specific reactivegroup or a photo-reactive group that will cause the scFv binding groupto permanently and covalently associate with the targeting scFv. Thephotoreactive group could be placed adjacent to the hapten or could bestructurally part of the hapten. A scFv that binds the hapten or themodified hapten may be created. The photoreactive hapten is called a“photohapten.” Illumination of the cell or tissue containing thephotohapten-linker-probe and the scFv-protein of interest would causecovalent linkage of the photohapten groups that are within the scFvbinding site to a region of the scFv. A potential advantage of thisapproach is that excess non-scFv associating hapten-linker-reportermolecule could be washed out, so long as it does not photochemicallyreact with other cellular structures. Any of a variety of knownphotoreactive groups may be used, such as those of the nitrene family.Several examples of reporter molecules are shown in FIG. 4. Thecompounds have sites for attachment to a linker and thus to a reportermolecule.

Variation 3: Photo-Controlled Reversible Binding of the ReporterMolecule to the scFv.

The binding of the reporter molecule may be reversibly controlled withlight by a photoreaction upon illumination of the scFv binding groupchromophores that alters its molecular conformation and thereby itsaffinity for the binding site of the targeting scFv. There are knownorganic chromophores that undergo conformational changes uponillumination. Stilbenes and azo-compounds undergo cis-transisomerization. Spiranes undergo ring opening as shown in FIG. 5.

In producing such a reversible system, the scFv may be generated usingthe predominant species of the equilibrium at room temperature, whichfor example is the spiro form of the compound in FIG. 5 that is >99% inthe absence of UV light. When the reagent system is in the cell ortissue, the reporter molecule would then be released from thescFv-protein of interest by illuminating the sample with UV light. Forrecapture of the reporter molecule by the scFv a longer wavelength ofillumination that excites the mero form may be used, or the reaction maybe incubated to achieve thermal re-equilibrium of the reaction to favorthe spiro form. Such a reagent system may be used to transient releaseof metabolic factors or drugs that modify regulatory pathways in cellsand tissues. A variety of photoreversible chromophores are known in theart and have been recently described in Sakata, et al., Proc. Natl.Acad. Sci. USA (2005) 102(13):4759-4764.

Variation 4: Photo-Release of Targeted Reporter Molecule

Photo-uncaging of cell and tissue modulating agents have been widelyused by biologists and biophysicists. Generally, an inactive form of amodulating agent or reporter molecule is illuminated to release anactive form into the illuminated region. In this case, the modulatingagent may be active, but targeted to the scFv-containing region of thecell or tissue. Illumination would release the material, allowing it todiffuse and produce effects elsewhere in the cell or tissue.Photo-uncaging chemistries are known (Curtin, et al. Photochemistry andPhotobiology (2005) 81:641-648) and may be inserted at a convenient sitebetween the hapten and the reporter molecule.

Currently, there is no way to target such reagents to specific types ofcells in a heterogeneous mixture of cells. Through genetic targeting,the reporter molecule could be sequestered and remain caged in a definedregion of the cell through the genetic targeting of the scFv to the cellof interest. Also, addition of the “caged reporter molecule” to thecells would allow the targeted scFv containing cell to specifically andstrongly bind the caged reporter molecule. Washing the cells wouldremove the caged reporter molecule from cells that are not of interestand illumination would produce release of the reporter molecule only inthe cells of interest.

In this invention scFv-based binding proteins offer one type offluorescent binding protein that can be used to create biosensors as inExample 3. As mentioned earlier there are other FBPs that could also beengineered to create sensors.

Example 3 A Biosensor for Protein-Protein Interaction Based on scFvTechnology

Protein-protein interactions are widely used by living cells to regulateimportant pathways controlling cell growth and behavior. There areexamples in the literature of the study of protein-protein interactionsby protein complementation (T K Kerppola (2006) Nature Methods 3:969).The protein-protein interaction event is detected by attaching the twocleaved parts of a sensor protein to the two proteins whose interactionis to be detected. When the interaction takes place the cleaved parts ofthe sensor protein complement (interact with) one another to form afunctional protein. The known examples in the literature do not includethe use of fluorescent reporter binding scFvs described above.

This example involves the use of the heavy (H) and light (L) fragmentsof single chain antibodies (scFvs) that have been selected to bindfluorescent reporter molecules. Once a ScFv with appropriate fluorogenhas been obtained and the genetic sequence of the ScFv determined,molecular biology methods are available to modify genetic sequences intothe ScFv gene at certain locations. The modified genetic sequences canbe inserted into yeast expression systems to produce secreted protein orto produce surface displayed ScFv that correspond to the geneticmodifications. It is further possible to eliminate the genetic sequencecorresponding to the short polypeptide linker that holds the H and Lchains of the selected ScFv together. Thus it is possible to obtainindependently the H and L halves of the original ScFv. It is furtherpossible to attach the genetic sequence of the H chain to a protein, forexample, P1, and the L chain to a second protein, P2 to obtain two DNAsequences that will give fusion proteins upon protein expression. Thegoal is to investigate whether P1 and P2 interact within or outsidecells. The genetic sequences for the P1-H and P2-L fusion proteins canthen be transfected into living cells where the cells will produce thetwo fusion protein products. In this example when the P1-H and P2-L arediffusing independently in the interior of a cell, neither the H nor theL fragments alone will bind the fluorogen (that bound with afluorescence increase to the original ScFv from which the H and L halveswere obtained) to produce a significant fluorescence signal. However,when P1 and P2 interact, the H and L components will be brought intoclose proximity and will interact as well to form the original combiningsite that will bind to the fluorogen. If this is the case, afluorescence signal will occur on interaction of P1 and P2.

The protein-protein biosensor of this example has advantages of quickfluorescence response, good sensitivity and relative reversibility. Itis possible to use fragments of other FBPs to create protein-proteininteraction sensors similar to the one described above.

Example 4 Eight Unique FBPs That Elicit Intense Fluorescence FromOtherwise Dark Dyes

In this experiment, it was demonstrated that fluorescent proteins forlive cell applications could be created. Eight unique FBPs that elicitintense fluorescence from otherwise dark dyes were isolated by screeninga yeast display library of human single chain antibodies (scFvs) usingderivatives of thiazole orange (TO) and malachite green (MG). Whendisplayed on yeast or mammalian cell surfaces, these FBPs bind theirfluorogens with nanomolar affinity, increasing their respective green orred fluorescence by several thousand-fold to brightness levels similarto that of enhanced green fluorescent protein. Significant spectralvariation is generated within the family of malachite green FBPs by useof different proteins and chemically modified fluorogens. These diverseFBPs and fluorogens provide opportunities to create new classes ofbiosensors and new homogeneous cell-based assays. These studies wereaimed at creating a new class of protein/dye reporters whose spectralproperties are determined by the interplay of a protein moiety (aFluorescent Binding Protein or FBP) and a noncovalently bound fluorogen.In the ideal case: (1) neither the FBP nor the fluorogen exhibitsfluorescence in the absence of the other, (2) the increase influorescence upon binding is dramatic, (3) the fluorogenic interactionbetween FBP and fluorogen is highly specific, eliminating the need forwashes or blocking agents, (4) neither the FBP nor the fluorogen istoxic or have intrinsic biological activity, 5) variation of the FBPand/or the fluorogen elicits useful variation in fluorescence color,binding affinity and other properties, and 6) the FBP can fold orreadjust its conformation rapidly with an associated fluorescencechange.

For example, such a system may allow the experimenter to modulatefluorescence as needed by adding or removing fluorogens. Or, fluorogenscould be tailored to address specific requirements, such as cellmembrane permeability and exclusion, or binding to a given FBP withdifferent affinities and colors to facilitate pulse-chase techniques.Unrelated fluorogen-FBP pairs that do not cross-react could be developedto support FRET applications. As proof-of-principle, it was demonstratedthat cells expressing single chain antibodies (scFvs) display intensefluorescence enhancement after exposure to two unrelated dyes, TO1 andMG. ScFvs were chosen because these small (<30 kDa) molecules retain thefull range of antigen recognition capabilities of the humoral antibodiesand are amenable to use as recombinant tags in fusion proteins. Acomplex human scFv library composed of ˜10⁹ synthetically recombinedheavy and light chain variable regions was available in a yeastsurface-display format, enabling us to use Fluorescence Activated CellSorting (FACS) to directly screen for fluorogenic binding to the dyes.

TO1 and MG are known fluorogens; strong fluorescence activation isobserved when TO1 intercalates into DNA or when MG binds to a specificRNA aptamer. Without being bound by theory, enhanced fluorescence isthought to occur because rapid rotation around a single bond within thechromophore is constrained upon binding. Enhanced fluorescence of such‘molecular rotors’ has also been reported for an antibody-dye complex,although with comparatively modest increase.

TO1 and MG were coupled to 3350 or 5000 MW polyethylene glycol(PEG)-biotin, and the dye-PEG-biotin conjugates were used withstreptavidin and anti-biotin magnetic beads to affinity enrich the yeastsurface display library for dye-binding scFvs. The TO1- and MG-affinityenriched scFv libraries were further enriched and then screened forfluorescence-generating scFvs by 2-4 rounds of FACS using the dye-PEGconjugates. For subsequent binding studies, TO1 and MG were coupled todiethylene glycol diamine (TO1-2p and MG-2p) to maintain antigenicstructure and aqueous dye solubility.

Sixteen clones that enhanced MG-2p fluorescence and two clones thatenhanced TO1-2p fluorescence were isolated from the library (FIG. 8).Sequence analysis revealed that the TO1-2p scFvs were encoded bydifferent heavy and light chain germline genes. The fluorogenic MG-2pscFvs represented six germline configurations, three composed of theusual heavy and light chain segments, and three composed of only asingle heavy or light chain segment (FIG. 9). The 11.5-14.4 kD singlechain species are about half the size of GFP (26.7 kD). When expressedon the yeast cell surface, spectra of the fluorescent scFvs could bedetermined in a 96 well homogenous assay format in the presence of freedye. We took advantage of the robust surface expression to spectrallycharacterize and determine the affinity of all of our FBPs when bound tothese two fluorogens (FIG. 9). The dissociation constants for HLI-TO1and HL2-TO1 were high nanomolar range. To obtain stronger binders, oneof the clones (HL1-TO1) was affinity matured by directed evolution usingtwo rounds of error prone PCR mutagenesis and FACS selection forincreased fluorescence at low fluorogen concentration, generatingseveral FBPs with improved properties (FIG. 8). The most improved FBP,HL 1.0.1-TO1, bound TO 1-2p with a cell surface K_(D) of about 3 nM. HL1.0.1-T01 and HL2-TO1 each exhibited modest red excitation shifts (509and 515 nm, respectively) relative to free dye absorbance (504 nm) butdiffered significantly from one another in emission maxima (530 and 550nm.)

Each of the anti-MG FBPs bound MG-2p tightly when assayed on the cellsurface, with an apparent K_(D) in the low nanomolar range. Cell surfacespectra showed significant variation among MG-2p binding FBPs.Excitation maxima of the FBP/MG-2p complexes ranged from 620-640 nm,markedly to the red of free dye absorbance (607 nm), whereas emissionmaxima ranged from 645-670 nm.

To more rigorously investigate the properties of FBP/fluorogens,secreted forms of HL1.0.1-TO1, HL4-MG and L5-MG were produced andaffinity-purified (FIG. 8). In solution HL1.0.1-TO1 bound TO1-2p with aK_(D) very similar to that observed on the cell surface. Directmeasurement showed that the fluorescence of TO1-2p increased about2.600-fold upon binding to the HL 1.0.1-TO1. The extinction coefficientand quantum yield of the HL1.0.1-TO1/TO1-2p complex (Σ=60,000 M⁻¹ cm⁻¹and Ø=0.47) are comparable to the values for Aequorea EGFP (53,000 and0.60), and predict that this FBP/fluorogen has EGFP-like brightness.

HL4-MG and L5-MG respectively showed 185- and 265-fold reduced affinityfor MG-2p in solution as compared to surface display but affinity ofH6-ME was reduced only five-fold (FIG. 9). This behavior differsmarkedly from that of HL1.0.1-TOL. The quantum yield of the HL4-MG/MG-2pcomplex (0.17) is similar to the quantum yield (0.187) of the malachitegreen RNA aptamer. Our result reflects a fluorescence enhancement ofabout 18,000-fold as compared to free fluorogen, which is much greaterthan the 40 to 100-fold enhancement for other antibody/fluorogencomplexes but less than the 50.000-fold increase observed when aquenched FlAsH-EDT2 reagent binds its cognate tetracysteine peptide.Absorbance of the FBP/fluorogen is more than 1.4-fold greater than freeMG-2p, corresponding to an extinction coefficient of about 105,000M⁻¹cm⁻¹. The combined absorbance and quantum yield predict a redfluorescent probe with high brightness. We have synthesized and obtainedseveral derivatives of the MG fluorogen to explore whether fluorogenicproperties can be usefully modulated by altered chemistry, and haveobserved among our six MG FBPS many changes in binding affinity,fluorescence intensity and excitation/emission spectra. FIG. 10Billustrates striking spectral changes produced by 3 MG fluorogenderivatives. It can be seen that a given fluorogen derivative tends toshift the spectrum to the blue or the red within a spectral contextestablished by the FBP. However, in some cases for some fluorogens,specific features of the FBP can greatly alter the extent of this shiftor generate new spectral bands. Malachite green and most derivativeshave a secondary absorbance peak at near ultraviolet to blue wavelengthsthat is sensitive to fluorogenic modulation by the FBP. We have takenadvantage of this behavior to demonstrate fluorescent reporting in twocolors excited by a single laser (non-overlapping green and red colorssingly excited by the 488 nm argon laser.) It was found that color is aproperty of the combined FBP/fluorogen. Protein context thus changes thefluorescent behavior of these fluorogens, as has also been shown forstilbenes.

It has been long established that certain dyes exhibit enhancedfluorescence upon binding to proteins, and there are other reports offluorogenic binding of dye to an antibody. Several features of what wereport are new and provide promise for development of a new generationof biosensor systems and live-cell assays. Unlike fluorogen-activatingmonoclonal antibodies previously described, the fluorogen-activatingscFvs described here are relatively small and compact monomeric proteinsthat can be recombinantly manipulated and expressed, making scFvs suitedfor use as genetically expressed tags or as injectable sensors. UnlikeGFP and related fluorescent proteins, the reversibly bound fluorogenchromophore is directly accessible to experiment and its chemistry canbe modified to alter reporting and sensing capabilities. In particular,control of access to the dye can afford a new level of selectivity.Unlike the biarsenical target peptide and the enzymatic peptide tags,scFvs provide a rich well-understood source of binding variation, whichcan be exploited to clone new FBPs that bind new fluorogens, or toenhance the functionality of existing FBPs and fluorogen variants usingdirected evolution technology.

These studies demonstrated that FBP/fluorogen reporters are well suitedfor expression at the extracellular side of the cell membrane. AlthoughscFvs are derived from extracellular antibodies and contain internaldisulfide linkages, it has been shown that functional scFvs can beexpressed intracellularly in a disulfide-free format. In addition, theFBP concept can be extended to protein scaffolds beyond scFvs, as hasbeen done for dye binding motifs such as the rhodamine bindingfluorettes and other structures.

Example 5 Materials and Methods for Example 5

Yeast Display Library

A yeast cell surface display library, composed of ˜10⁹ recombinant humanscFv's derived from cDNA representing a naive germline repertoire, isobtained from Pacific Northwest National Laboratory (PNNL). The originalversion of the library was obtained from PNNL and was the source of ourfirst isolated FBP, HL1TO1. However, this library was subsequently foundto be contaminated by a low level of another yeast strain (Candidaparapsilosis) that overwhelmed yeast cultures afterrepeated outgrowthsteps. We obtained another library that represents a subset of theoriginal PNNL library. The estimated complexity of this library is˜8×10⁸ independent scFvs. This library shows no evidence ofcontamination, and was the source of all other FBPS isolated for thisstudy. This uncontaminated library version is currently available fromPNNL.

Yeast Strains

EBY100 was host to the yeast display library and YVH10 was used tosecrete scFvs, as described. For studies of individual FBPs, pPNL6plasmids were transferred to JAR200, a G418 resistant derivative ofEBY100. JAR200 expressed higher levels of displayed scFvs and gavehigher transformation transformation rates with pPNL6 plasmids thanEBY100.

-   YVH10: Mat α ura3-52, trp1, leu2δ200, his3δ200, pep4:HIS3, prbd1.6R,    can1, GAL, GAPDH promoter-PDI1-   EBY100: Mat α ura3-52, trp1, leu2δ200, his3δ200, pep4:HIS3,    prbd1.6R, can1, GAL, GAL promoter-AGAI:URA3-   AR200: Mat α ura3-52, trp1, leu2δ200, his3δ200, pep4:HIS3, prbd1.6R,    can1, GAL, GAL promoter-AGAI::URA3:G418r    Yeast Buffer System

We employed a modified PBS buffer (PBS pH 7.4, 2 mM EDTA, 0.1% w/vPluronic F-127 (Molecular Probes, Invitrogen)) for magnetic beadenrichment, FACS experiments, and all assays of yeast surface displayedor purified scFvs. Inclusion of Pluronic F-127 was found to greatlyreduce absorption of low concentrations of TOI and MG dyes to plasticand glass surfaces; microplate samples of 500 nM free dye gave stablereadings for at least 18 hours.

Standard Procedure for Cloning of Single Chain Antibodies

All FBPs other than HL1-TOI (see below) were cloned essentially asdescribed except that a 2-color FACS enrichment screen based on enhancedfluorescence of the fluorogen was employed instead of a 2-color screenbased on antigen labeled with independent fluorophore. FACS enrichmentwas carried out on a Becton Dickinson FACSVantage SE with FACSDivaoption; candidate FBPS were autocloned onto agar plates prior tocharacterization. 1 μM TO1-PEG5000-biotin or 500 nM MG-PEG5000 biotinwere used to magnetically enrich and sort for respective FBPS, exceptthat 50 nM MG-PEG5000-biotin was used to autoclone highest affinityMG-FBP candidates.

Cloning of HL1-TOI

A large population of induced cells was directly enriched for FBPS by 3successive rounds of FACS. Briefly, cells were enriched for affinity bytwo rounds of magnetic bead treatment, and the output cells grown andinduced. 10⁸ of these cells were sorted on a MoFlo high speed FACS, andthe output 9×10⁶ cells were immediately resorted under the sameconditions to give 7×10⁴ cells. These cells were again sorted to give˜1500 cells as final output. After growth and induction, these cellswere sorted on an Epics Elite FACS, and the small population of cells(˜0.5%) with significantly improved fluorogen signal was collected,regrown and resorted. These cells exclusively displayed HL1-TO1.Subsequent attempts at cloning other FBPs using this direct approachfailed.

Identification of FBPs

Autocloned yeast cells displaying candidate FBP isolates were grown insmall cultures, and yeast plasmid DNA isolated using a Zymoprep kit(Zymo Research). The scFv insert was PCR amplified, and the amplifiedDNA product purified on an agarose gel and then DNA sequenced. scFvvariable region sequences were classified as to human germlinecomposition by analysis on the IMGTN-QUEST website.

Spectral Characterization of FBPs

Yeast surface displayed scFvs were spectrally characterized usingfluorescence bottom reading in 96 well microplates on a Tecan Safire2plate reader. 10⁶ cells in 200 μl yeast buffer were assayed with100-1000 nM MG-2p or TO1-2p. Spectra were corrected by subtraction offluorescence of control cells not expressing scFvs.

Affinity Maturation of HL1-TO1

Affinity maturation of HL1-TO1 followed described methods for randommutagenesis and selection of improved clones, except that the 2-colorFACS screen used in our standard cloning procedure was employed usingTO1-2p as the fluorogen.

Secretion and Purification of Soluble FBPs

Induction and secretion of scFvs were at 20° or 25° C. as described,except that YEPD was replaced by a tryptone-based secretion medium:

-   5 g/L casamino acids (-ade, -ura, -trp) 50 g/L Bacto-Tryptone (BD    #211705)-   1.7 g/L Yeast Nitrogen Base w/out ammonium sulfate amino acids (BD    #233520)-   5.3 g/L ammonium sulfate-   10.19 g/L Na₂HPO₄.7H₂O-   8.56 g/L NaH₂PO₄.H₂O

FBP secretion in 1 liter cultures was monitored during the 2-5 daycourse of induction by assaying the fluorescence of culture supernatantson a 96-well Tecan Safire2 plate reader. 100 μl of 2× assay buffer (100mM Na_(x)H_(x)PO₄, pH 7.4, 4 mM EDTA, 0.2% Pluronic F-127) containingeither 1 μM TO1-2p or 200 nM MG-2p was added to 100 μl of culturesupernatant for reading; readings were corrected for background bysubtracting the fluorescence of identically treated virgin secretionmedium. Tryptone secretion medium gave 2 to 10-fold increased yields ofsecreted scFvs as compared to YEPD.

Culture supernatants were dialysed and concentrated 3 times against 6liters PBS on an Amicon Model 2000 high performance ultrafiltration cellusing a 10,000 mw cut-off cellulose membrane. To purify the 6-his taggedFBPS, the concentrated dialysate (˜50 ml) was subjected tonickel-nitrilotriacetic acid chromatography (Ni-NTA) according tomanufacturer's instructions. Appropriate dilutions of eluted fractionswere assayed for fluorogenic activity using essentially the same assayas for secretion. Fluorescent fractions were pooled, assayed for proteincontent using a BCA protein assay kit, and analyzed by SDS gelelectrophoresis.

Determination of Fluorogen Binding Affinity to Yeast Surface DisplayedFBPs

A homogenous assay under equilibrium binding conditions was devised todetermine the binding affinity of fluorogen to yeast displayed scFvs. Aflow cytometric method for titrating yeast displayed scFvs withfluorescently labeled antigen was adapted to the use of fluorogens. 10⁶induced yeast in 200 μl modified PBS (˜1 nM displayed scFvs) containingfluorogen over a concentration range of 0.1-1000 nM were assayedin_duplicate for fluorescence in 96 well microplates on a Tecan Safire2reader. As controls,_mock induced JAR200 cells that do not express scFvswere treated with equal concentrations of fluorogen; fluorescence wascorrected by subtraction of the fluorescence of control cells. Cellsurface K_(D) values were determined on Prism Graphpad Prism 4.0software by non-linear regression analysis using a one-site bindingalgorithm for saturation binding:Y=B _(max) *X=(KD+X)where X is the concentration of fluorogen.Determination of Fluorogen Binding Affinity to Soluble FBPs

Binding affinity to soluble scFvs was determined by monitoringfluorogenic signal under conditions of ligand depletion using ahomogenous 96 well microplate assay similar to above. 1 nM HL1.0.1-TO1,10 nM L5-MG and 100 nM HL4-MG were each assayed with a 0.1 to 1000 nMrange of fluorogen. Fluorescence of each FBP+dye sample was corrected bysubtracting the fluorescence of a dye only sample. K_(D) values weredetermined by non-linear regression using Graphpad Prism 4.0 and aligand depletion algorithm.Y=(X+K _(D) +R−√{square root over ((−X−K _(D) −R)²−4*X*R)})÷2where X is the concentration of fluorogen, and R is the concentration ofFBP/fluorogencomplex at the observed or extrapolated plateau at maximumfluorescence.Determination of Quantum Yields

Quantum yields were determined by comparing integrated spectra ofFBP/fluorogen complexes with those of reference dyes. Corrected emissionspectra were taken at concentrations of FBP/fluorogen complex andreference dyes giving similar absorbances at the excitation wavelength,and the intensity integral computed. Provisional quantum yields werecalculated by the relation:

$\Phi = {\Phi_{R}*\frac{I*A_{R}*\eta^{2}}{I_{R}*A*\eta_{R}^{2}}}$where (phi is the quantum yield, I is the integrated intensity, A is theabsorbance, η is the refractive index, and R designates the referencedye. Provisional quantum yields were adjusted to 1:1 complexation byusing the solution K_(D) of the complex to quantify the proportionaloccupancy at these concentrations (using Graphpad Prism 4.0 and theabove ligand depletion algorithm).

A cyanic dye, Cy5.18 in PBS and Di-S—C2-(5) in MeOH were used asreference fluorophores for the determination of HL4-MG/MG-2p andL5-MG/MG-2p quantum yields. 2 μM scFv and 440 nM MG-2p were employed;emission spectra of respective complexes were taken in duplicate.Absorbances of the respective complexes were significantly higher thanfree MG-2p. These differences in absorbance magnitude underrepresentactual differences because of incomplete complexation at theseconcentrations. HL4MG/MG-2p and L5-MG/MG-2p quantum yields wererespectively multiplied by 1.35 and 1.20 to correct for incompletecomplexation.

Fluorescein in 0.1 N NaOH and Rhodamine-6-G in MeOH were used asreference fluorophores for the determination of the quantum yield ofHL1.0.1-TO1/T012p. At the employed concentrations of 2 μM scFv and 520nM TO1-2p, virtually complete complexation is expected, and nocorrection was used. Only slight changes in absorbance magnitude werenoted.

Determination of Fluorogenic Enhancement

Fluorogenic enhancement of HL4-MG, L5-MG and HL1.0.1-TO1 was measured in96 well microplates on a Tecan Safire2 reader by comparison of thefluorescence of 500 nM free fluorogen with the fluorescence of a mixtureof 500 nM fluorogen and 2 μM respective FBP. After a 1 hour incubationto allow complex formation, fluorescence was measured at the excitationand emission maxima of the fluorogen/FBP complexes. Fluorescence ofyeast PBS buffer was subtracted from all samples. Fluorescence readingswere stable for at least 16 hours.

Extended gain settings were used to increase numerical accuracy. ForHL4-MG and L5MG, the differences in fluorescence between complexes andfree dye exceeded the extended gain range of the instrument, sointermediate concentrations of Cy5 were also measured to allowinterpolation between the extreme values. Two independent experiments,each with triplicate measurements, were carried out for each of the 3FBPS; standard deviations for averaged values of FBPS, free fluorogen orPBS were less than 5%. The fold-enhancement values for HL4-MG and L5-MGwere respectively multiplied by 1.35 and 1.20 to correct for partialcomplexation.

Mammalian cell-surface expression of FBP molecules

Plasmids expressing surface-displayed scFv's were generated as follows.A 375 by PCRamplicon was amplified from E. coli C600 DNA using asprimers:

SEQ ID NO: 23: GGGGCTACCAGTTTGAGGGGACGACGA SEQ ID NO: 24:GGCCCCTGCGGCCGTTAGCTCACTCATTAGGCA

This molecule, which contains the lac promoter and 271 nucleotides ofbeta-galactosidase coding sequence flanked by SfiI sites, was cut withXmaI and ligated into pDisplay (Invitrogen) between the Smal and XmaIsites to produce vector pDisplayBlue. Individual scFv sequences wereprepared for insertion between the SfiI sites in pDisplayBlue byPCR-amplifying the scFv sequences from pPNL6 clones using as primers:

SEQ ID NO: 25: TATATAGGCCCAGCCGGCCTACCCATACGACGTTCCAGAC SEQ ID NO: 26:TATATAGGCCCCTGCGGCCAATTCCGGATAGGACGGTGAG

These amplicons were cut with SfiI, ligated into SfiI-cut vector,transformed into DHScc E. coli to ampicillin resistance, and Lac+colonies picked for DNA sequencing. DNA was prepared from selectedtransformants using Qiagen Mini-Prep kits and transfected into NIH3T3cells or M21 mouse melanoma cells (˜1 μg DNA per 10⁵ cells) in24-wellplates using Lipofectamine 2000 (Invitrogen) following theprotocols supplied by the manufacturer. Stable transfectants wereisolated by successive rounds of FACS sorting of cells exposed to theappropriate fluorogens.

Example 6 Fusion of scFv Sequences to a Transmembrane Domain Derivedfrom Human Fibroblast Growth Factor Receptor 2, and Expression of theFusion Constructs in Mammalian Cells

Mammalian Cell-Surface Expression of Fluorogen-Activating PolypeptideMolecules.

NIH3T3 cells stably expressing HL4-MG fused to PDGFR were imaged byconfocal microscope at 633 nm excitation after treatment for 5 min inPBS with 200 nM MG-11p or 200 nM MG-ester. On longer incubation,MG-ester illuminated intracellular features such as the nuclearperiphery (endoplasmic reticulum) and Golgi become more difficult tovisualize. Fluorescence images were excited at 633 nm using a 650 nmlong pass filter. Fluorescence images were unprocessed; interferencelines on DIC images were removed using a fourier transform filter inPhotoshop. Images in were acquired on a Zeiss LSM510 META laser scanningmicroscope using a 63× objective. Fluorescence images were excited at633 nm using a 650 nm long pass filter.

In addition, surface labeling of human tumor cells with a MGfluorogen-activating polypeptide was shown. Stably transformed M21melanoma cells expressing HL4-MG fused to PDGFR were imaged as aconfocal stack at 488-nm excitation using 10 nM MG-11p. Images wereacquired on a Zeiss LSM510 META laser scanning microscope using a 63×objective. Images were acquired on a Zeiss Axioplan 2 with Apotomemicroscope. Green false color (TO1-2p) was imaged using 540/25 and605/55 nm excitation and emission filters; red false color was imagedusing 560/55 and 710/75 nm excitation and emission filters. Images werereconstructed from 72 1 μm sections, displayed as 15 projections in NIHImage software, and false colored in Adobe Photoshop.

Also, surface labeling of fibroblasts with a TO1 fluorogen-activatingpolypeptide was detected. Stably transformed NIH3T3 cells expressingHL1.1-TO1 fused to PDGFR and imaged using 40 nM TO1-2p. Images wereacquired on a Zeiss Axioplan 2 with Apotome microscope. Green falsecolor (TO1-2p) was imaged using 540/25 and 605/55 nm excitation andemission filters; red false color was imaged using 560/55 and 710/75 nmexcitation and emission filters. Images were reconstructed from 72 1 μmsections, displayed as 15 projections in NIH Image software, and falsecolored in Adobe Photoshop. The HL1.1-TO1 expressing cells were excitedat 488 nm and visualized with a 500 nm long pass filter.

In another experiment, it was shown that simultaneous surface labelingof fibroblasts with MG and TO1 FAPs occurred. NIH3T3 cells respectivelyexpressing the FAPs 1:1 and imaged using 10 nM MG-2p and 40 nM TO1-2p.The transparency of surface-labeled cells allows fine discrimination ofcontact surfaces between cells of different colors. Images were acquiredon a Zeiss Axioplan 2 with Apotome microscope. Green false color(TO1-2p) was imaged using 540/25 and 605/55 nm excitation and emissionfilters; red false color was imaged using 560/55 and 710/75 nmexcitation and emission filters. Images were reconstructed from 72 1 μmsections, displayed as 15 projections in NIH Image software, and falsecolored in Adobe Photoshop. a 700/75 nm bandpass filter was used tovisualize the 488 nm excitation of HL4-MG

Example 7 Fusion of scFv Sequences to the Human Glucose TransporterGLUT4, and Expression of the Fusion Constructs in Mammalian Cells

An open reading frame comprising the coding sequence of human GLUT4 wascloned 5′ to the PDGFR sequence in the modified pDisplay vectordescribed in Example 5 that was further modified to accept the insertbetween PflMI restriction sites. Fluorogen-activating scFv sequenceHL1.1-TO1 was cloned between the SfiI sites of these constructs asdescribed in Example 5. NIH3T3-L1 cells were transfected with theconstructs, and stable transfectants were isolated by multiple rounds ofFACS sorting for fluorogen-activating cells.

Imaging by fluorescence microscopy after incubation in the presence offluorogen showed distinct surface labeling when cells were treated witha membrane impermeant fluorogen, and internal as well as surfacelabeling when cells were transfected with a control plasmid with EGFPcloned between the SfiI sites.

Images were taken with Apotome microscope with 63× water immersion lens.Excitation occurred at 488 nm with emission collected in GFP channel.Cells are undifferentiated 3T3-L1 fibroblasts. These results indicatethat a fusion protein between GLUT4 and a fluorogen-activating scFv iscorrectly localized at the cell surface, as well as in the expectedsubcellular compartments of the endomembrane system.

Example 8 Fusion of scFv Sequences to the Human G-Protein CoupledReceptor ADRB2, and Expression of the Fusion Constructs in MammalianCells

The coding sequence of human ADRB2 was PCR amplified from an ADRB2fosmid and cloned into the BsmI site in the modified pDisplay vectordescribed in example N, with an in frame stop codon at the 3′ end of theADRB2 sequence. Fluorogen-activating scFv sequences HL1.1-TO1 and HL4MGwere cloned into the SfiI sites of these constructs as described inexample N. NIH3T3 cells were transfected with the constructs, and stabletransfectants were isolated by multiple rounds of FACS sorting forfluorogen-activating cells.

Imaging by fluorescence microscopy after incubation in the presence offluorogen showed distinct surface labeling when cells were treated withmembrane impermeant fluorogens, and internal as well as surface labelingwhen cells were treated with membrane permeant fluorogens. Further, whencells were treated with the ADRB2 agonist isoproterenol at physiologicalconcentrations, internalization of the ADRB fusion protein was observed,indicating that the fusion protein was physiologically active withrespect to recognition and internalization of the agonist.

Example 9 Cell Surface Complementation

This experiment demonstrated that the light chain and heavy chains of aselected scFv can be separately fused to two different proteins, andthat when these two proteins are in close proximity, the heavy and lightchains associate to produce a binding site for the same dye that bindsto the original scFv leading to a fluorescence increase. In thefollowing experiments three classes of yeast cells were generated bymolecular biology methods known in the art. One class of yeast expressedonly the heavy chain of the scFv on the surface. A second classexpressed only the light chain of the scFv on its surface. The thirdclass expressed both the heavy and light chains on the surface. When thefluorescent reporter TO1 was added independently to solutions containingthe three classes of yeast cells, only the third class that expressedboth heavy and light chains at a high surface density produced afluorescence increase, as shown below. Specifically, two vectors, pPNL6and pPNL6URA3 were prepared, where the TRP1 gene of pPNL6 was replacedwith the URA3 gene of S. cerevisiae. This approach allowed selection forboth plasmids in a single cell. Fragments carrying scFv1, scFv1 HO(heavy only chain), or scFv1 LO (light only chain) were cloned into eachof the two vectors.

JAR200 yeast cells were transformed with each single plasmid as well asboth scFv1 HO (PNL6)+scFv1 LO (PNL6URA3) or both scFv1 HO(PNL6URA3)+scFv1 LO (PNL6). Analysis by FACS and TECAN followedinduction of the cells. In both cases, 1 μM TO1-2P was used.

In the first approach, flow cytometry was used to assay cellularfluorescence of the three classes of yeast in the presence and absenceof TO1. The population of induced cells was analyzed by measuringfluorescence emitted at 685 from a signal caused by protein labeled byanti-C-myc Alexa 647. This allowed confirmation that the three classesof yeast cells were expressing full length heavy, light, or heavy andlight chains on the cell surface. For cells that expressed full lengthfragments the TO1-2p fluorescence was observed as emission at 530.

TABLE 1 PLASMID MEAN 530 SIGNAL scFv1 PNL6 840 Intact origininal scFvscFv1 PNL6URA3 760 Intact origininal scFv HO PNL6 88 Heavy chain only HOPNL6URA3 82 Heavy chain only LO PNL6 102 Light chain only LO PNL6URA3100 Light chain only HO PNL6 + LO 317 Heavy and light same cell PNL6URA3HO PNL6URA3 + 261 Heavy and light same cell LO PNL6

The cells expressing both heavy and light were 2.5-3 times morefluorescent than cells expressing heavy or light only. The signalincrease is likely to be larger in assays where the two proteinscontaining the heavy and light chains are associating in a pair-wisemanner rather that a statistical association. With pair-wiseprotein-protein interactions the fluorescence of the complex mayapproach that of the original scFv PNL6 or PNL6URA3 in which the heavyand light chains are directly linked through ashort-serine-glycine-polymeric linker.

In the second approach, a fluorescence spectrometer was used to quantifythe fluorescence from suspensions of cells. The fluorescence excitationwavelength was 506 nm and the emission wavelength was 610 nm. Valuespresented below are corrected from raw data by subtracting out thevalues for (buffer+dye) and induced (no dye) samples. They were thencorrected for the percent of the population that was induced, asdetermined by the flow cytometry analysis analysis.

TABLE 2 F/(% Pop PLASMID Fluorescence % Pop. Ind IND) scFv1 PNL6 2393460.3 39691 scFv1 PNL6URA3 28686 48.4 59268 HO PNL6 135 84.2 160 HOPNL6URA3 −605 70.6 / LO PNL6 −16 73.7 / LO PIML6URA3 882 64.4 1369 HOPNL6 + LO PIML6URA3 6553 84.2 7782 HO PNL6URA3 + LO PNL6 4571 73.1 6253

The fluorescence spectroscopy measurements demonstrated that cellsexpressing both heavy and light were significantly more fluorescent thancells expressing heavy or light only.

Example 10 Syntheses of Dyes

2-[(1-[3-[3[(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)propyl]-4(1H)quinolinylidene)methyl]-3-(3-sulfopropyl)benzothiazoliniuminner salt 3

1-(3-N-Phthalimidopropyl)-4-methyl-quinolinium bromide 2 (822 mg, 2mmol) and 3-(3-Sulfopropyl)-2-methylthio-benzothiazole 1 (1.2 g, 4 mmol)were dissolved in 150 mL boiling anhydrous ethanol. Triethylamine (0.28ml, 4 mmol) was gradually added over a 15 minutes time period. Thereaction mixture was refluxed for one hour. The precipitated solid wasfiltered off from the hot reaction mixture to give 822 mg of a redpowder. Yield: 820 mg (70%); C₃₁H²⁷N₃O₅S₂MW=585.7 g/mol

Theory: C 63.57% H 4.65%; N 7.17% Found: C 63.62% H 4.61% N 7.05%

¹H-NMR (CDCl₃/MeOD): 8.85 (1H, d); 8.33 (1H, d); 7.68-7.87 (8H, m);7.52-7.62 (2H, m); 7.31 (2H, m); 7.06 (1H, s); 4.75 (2H, t); 4.51 (2H,t); 3.84 (2H, t); 3.11 (2H, t); 2.34 (4H, m).

-[(1-(3-Aminopropyl)-4(1H)-quinolinylidene)methyl]-3-(3-sulfopropyl)benzothiazoliniumhydrochloride “TO1” 4

2-[(1-[3-[3[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)propyl]-4(1H)quinolinylidene)methyl]-3-(3-sulfopropyl)benzothiazolinium inner salt 3(585.7 mg; 1 mmol) was suspended in 20 mL concentrated hydrochloric acidand 5 mL ethanol. The reaction mixture was refluxed for 48 hrs. Thesolvent was removed under vacuum. The residue was dissolved in 10 ml, ofmethanol and the dye was precipitated with 50 mL methylene chloride. Thedye was filtered off; dissolved in water and filtered. The filtrate wasconcentrated under vacuum to give 400 mg of a red powder. Yield: 394 mg(80%) C₂₃H₂₆ClN₃O₃S₂ MW: 492.06 g/mol; Theory: C 54.16% H 5.53% N 8.23%;Found: C 54.39% H 5.70% N 7.82%

¹H-NMR (D₂O): 7.64 (1H, d); 7.63 (1H); 7.53 (1H); 7.43 (1H); 7.23 (1H);7.1 (1H); 6.92 (1H); 6.9 (1H); 6.54 (1H); 6.29 (1H); 5.9 (1H); 3.7 (4H);2.95 (2H); 2.82 (2H); 1.81 (4H). C₂₃H₂₆ClN₃O₃S₂ x H₂O; MW 510.07 g/mol;LTV/VIS λmax=488 nm; εmol, =58000

[2-[(1-(Boc-18-amino-4,9-diaza-12,15-dioxa-5,8-dioxo-octadecanyl)-4(1H)quinolinylidene)methyl]-3-(3-sulfopropyl)benzothiazolinium]inner salt “TO1-2P—NHBOC” 6

Boc-14-amino-5-aza-8,11-dioxa-4-oxo-tetradecanoic acid 5 (700 mg, 2mmol) was dissolved in dry CH₃CN (10 mL). N-Hydroxysuccinimide (2.1mmol, 300 mg) and dicyclohexylcarbodiimide (500 mg, 2.1 mmol) wereadded. The reaction mixture was stirred at room temperature overnight.The precipitated urea was filtered off and the filtrate was used in thenext reaction step. TO1-amine (500 mg, 1 mmol) was dissolved in amixture of water (10 mL) and acetonitrile (10 mL). The active estersolution was added in portions of 0.5 mL. After 30 min of stirring aprecipitate formed. TLC control (silica gel) showed the product in thesolution phase (9:1 CHC13/McOH/1 TFA). The solid was filtered off andthe filtrate concentrated under vacuum. The residue was purified bycolumn chromatography on silica gel. Yield: 1.1 g (75%); C₃₃H₄₄ClN₅O₇S₂MW: 725.48 g/mol 1H-NMR (MeOD): 8.64 (1H, d); 8.31 (1H, d); 7.75 (2H,m); 5.59 (2H, m); 7.44 (2H, m); 7.12 (1H, t); 7.07 (1H, d); 6.79 (1H,s); 4.65 (2H, bt); 4.39 (2H, t); 3.60 (4H, s); 3.5 (4H, m); 3.38 (2H,t); 3.21 (2H, t); 3.13 (2H, t); 2.58 (4H, m); 2.33 (2H, m); 2.06 (2H,m); 1.42 (9H, m). UV/VIS,% m, =508 nm

[2-[(1-(18-amino-4,9-diaza-12,15-dioxa-5,8-dioxo-octadecanyl)-4(1H)-quinolinylidene)methyl]-3-(3-sulfopropyl)benzothiazolinium]trifluoroacetate“TO1-2P”7

TO1-2P-BOC 6 (368 mg, 0.5 mmol) was dissolved in methanol (50 mL) and 1Nhydrochloric acid 5 mL was added. The reaction mixture was stirredovernight at room temperature. The solvent was removed under vacuum togive an orange resin. The product was purified by HPLC; Watersμ-Bondapak C18; gradient 10-40% water/acetonitrile/0.1% TFA¹H-NMR(MeOD): 8.53 (1H, d); 8.27 (1H, d); 7.75 (1H, t); 7.68 (1H, d); 7.55(1H, t); 7.49 (1H, d); 7.43 (1H, d); 7.37 (1H, t); 7.05 (1H, t); 7.01(1H, d); 6.68 (1H, d-exchanges); 4.57 (2H, t); 4.35 (2H, t); 3.72 (2H,t); 3.65 (4H, m); 3.57 (2H, t); 3.39 (2H, t); 3.31 (2H, t); 3.14 (2H,m); 3.12 (2H, m); 2.26 (2H, m); 2.04 (2H, m); C₃₅H₄₄F₃N₅O₉S₂ MW: 799.9g/mol εmol (λ504 in H2O) 34000; εmol (λ504 in MeOH) 49500

Biotin-PEG5000-TO1 9

2-[(1-(3-Aminopropyl)-4(1H)-quinolinylidene)methyl]-3-(3-sulfopropyl)benzothiazoliniumhydrochloride TO1 4 (5.8 mg; 0.01 mmol) was dissolved in 0.1 mL ofwater. Biotin-PEG5000-NHS ester 8 (Nektar Therapeutics) (50 mg; 0.01mmol) dissolved in 0.1 mL of DMF was added to the TO1 solution followedby 0.1 mL of saturated sodium bicarbonate solution. The reaction mixturewas stirred for 1 hr. The solvents were removed under vacuum and theresidue was taken up in a minimum of water and passed through a P4 sizedexclusion column to remove free TO1. The PEG fraction was concentratedand purified on Q-Sepharose (Amerscham Biosciences) to separateunlabeled Biotin-PEG from Biotin-PEG5000-TO1. MS: M_(n) 5743.56;

[4-(1-Oxa-3-carboethoxypropyl)phenyl]bis[4-(dimethylamino)phenyl-methane12

Dimethylaniline 10 (7.27 g; 60 mmol) and ethyl4(4-formylphenoxy)butanoate 11 (7.08 g; 30 mmol) were dissolved inanhydrous ethanol (300 mL). Anhydrous zinc chloride (8.2 g) was addedand the reaction mixture was refluxed for 2 days, distilling off theethanol 4-5 times and replacing it with anhydrous ethanol. After coolingto room temperature, the reaction mixture was concentrated. The residuewas taken up in ethyl acetate and water. The organic phase wasseparated, washed with water, dried, concentrated and purified on silicagel. Eluent: Ethyl acetate. MW C₂₉H₃₆N₂O₄ 476.62 g/mol; yield: 11.4 g(80%); ¹H-NMR (CDCl3): 7.1 (2H, d); 7.05 (4H, d); 6.85 (2H, d), 6.75(2H, d); 5.4 (1H.s, OH); 4.25 (2H, q); 4.05 (2H, t); 2.95 (12H, s); 2.6(2H, t); 2.25 (2H, m), 1.35 (3H, t)

Methylium,bis[4-(dimethylamino)phenyl](4-(3-carboethoxypropyl)phenyl)-chloride 13

[4-(1-Oxa-3-carboethoxypropyl)phenyl]bis[4-(dimethylamino)phenyl-methane12 (460 mg, 1 mmol) was dissolved in 25 mL ethyl acetate.Tetrachloro-p-benzochinone (490 mg/2 mmol) was added and the reactionmixture was refluxed for 1 hr. The reaction mixture was cooled to roomtemperature. Ethyl acetate (75 mL) was added and the product wasextracted with water (5×50 mL). The combined aqueous phase was washedwith ethyl acetate (2×50 mL) and concentrated to give 200 mg (40%) ofproduct. MW C₂₉H₃₅ClN₂O₄ 511.1 g/mol ¹H-NMR (CD3CN): 7.33 (4H, d); 7.08(2H, d); 6.92 (4H, d); 4.15 (2H, t); 4.10 (2H, q); 3.23 (12H, s); 2.48(2H, t); 2.08 (2H, m); 1.21 (3H, t)

[4-(1-Oxa-3-carboxypropyl)phenyl]bis[4-(dimethylamino)phenyl-methane 14

[4-(1-Oxa-3-carboxypropyl)phenyl]bis[4-(dimethylamino)phenyl-methane 12(5 g, 10.5 mmol) was dissolved in acetone (30 mL). Sodium hydroxide (10mL of a 2N aqueous solution) was added. The reaction mixture was stirredat room temperature until the ester was cleaved (TLC control). Theacetone was removed and the aqueous solution adjusted to pH 3-4 with 1 NHCl. The aqueous phase was extracted with ethyl acetate (3×50 mL). Thecombined organic phase was washed with water and brine and dried oversodium sulfate. The solvent was removed to give 4 g of a light greenresin (Yield: 90%). The compound was used without further purification.MW:C₂₇H₃₂N₂O₄ 448.6 g/mol. ¹H-NMR (CDCl3): 6.96 (2H, d); 6.88 (4H, d);6.81 (2H, d); 5.24 (1H, s); 3.92 (2H, t); 2.83 (12H, s); 2.34 (2H, t);1.91 (2H, m)

[4-(Boc-9-amino-6-aza-1-oxa-5-oxo-nonyl)phenyl]bis[4-(dimethylamino)phenylmethane15

[4-(1-Oxa-3-carboxypropyl)phenyl]bis[4-(dimethylamino)phenyl-methane 14(4.1 g, 9.14 mmol) was dissolved in a mixture of dry THF/CH₂Cl₂.Triethylamine (1.4 mL, 10 mmol) was added. The reaction mixture wascooled to 0° C. Ethyl chloroformate (0.95 mL, 10 mmol) was added and thereaction mixture was stirred for 30 min (TLC) control.NBOC-ethylenediamine (1.6 g, 10 mmol) was added. The reaction mixturewas stirred for 30 min at room temperature (TLC control silica gel,ethyl acetate). The solvent was removed and the residue was taken up inethyl acetate. The organic phase was washed with diluted sodiumbicarbonate, water and brine and dried over sodium sulfate. The solventwas removed under vacuum and the residue purified by columnchromatography on silicagel; eluent: ethyl acetate (RF 0.2). Light bluecrystals were obtained. MW C₃₄H₄₆N₄O₄ 574.8 g/mol. Yield: 4.2 g (80%).¹H-NMR (CD3CN): 7.01 (2H, d); 6.94 (4H, d); 6.82 (2H, d); 6.69 (4H, d);6.59 (1H, s, CONH); 5.59 (1H, s, CONH); 5.28 (1H, s, OH); 3.95 (2H, t);3.21 (2H, m); 3.11 (2H, m); 2.88 (12H, s); 2.28 (2H, t); 1.99 (2H, m);1.4 (9H, s, BOC)

[4-(9-amino-6-aza-1-oxa-5-oxo-nonyl)phenyl]bis[4-(dimethylamino)phenyl-methane16

[4-(Boc-9-amino-6-aza-1-oxa-5-oxo-nonyl)phenyl]bis[4-(dimethylamino)phenylmethane(118 mg, 0.2 mmol) was dissolved in HCI/ethanol (2 mL of a 5% solution).The reaction mixture was kept overnight at room temperature. The solventwas removed and the residue dried. The product was used as such in thenext reaction step.

[4-(Boc-9-Aminoethyl-PEG2)-6,9,15-triaza-1,12-dioxa-5,10,14-trioxopentaundecanyl)phenyl]bis[4-(dimethylamino)phenyl-methaneLeuko-MG-2P 17

[4-(9-Amino-6-aza-1-oxa-5-oxo-nonyl)phenyl]bis[4-(dimethylamino)phenyl-methane(118 mg, 0.2 mmol) 16 was reacted with the NHS-ester ofboc-[4-amino-5-aza-8,11 dioxa-4-oxo-tetradecanoic acid 5 in 1 DMF (1mL)/1N NaHCO₃ (1 mL). The reaction mixture was concentrated and purifiedby chromatography on silicagel (eluent: ethyl acetate/10-30% methanol).MW: C₃₄H₄₆N₄O₄ 574.8 g/mol

[4-(9-Aminoethyl-PEG2)-6,9,15-triaza-1,12-dioxa-5,10,14-trioxo-pentaundecanyl)MG-2P18

Leuko-MG2 17 (161 mg/0.28 mmol) was dissolved in 25 mL of ethyl acetate.Tetrachloro-p-benzochinone (100 mg/0.4 mmol) was added and the reactionmixture was refluxed for 1 hr. The reaction mixture was concentrated to2 mL. Hydrochlorid acid (2 mL of a 1N solution) was added and thereaction mixture was stirred at room temperature for 1 hr. The reactionmixture was partitioned between 100 mL ethyl acetate and 150 mL ofwater. The water phase was separated and washed with ethyl acetate(2×100 mL). The aqueous phase was concentrated. The residue was dried at60° C. under high vacuum to give 130 mg of dye (yield: 88%). MW:C₂₉H₃₇N₄O₂C1 509.1 g/mol ¹H-NMR (MeOD): 7.42 (4H, d); 7.36 (2H, d); 7.17(2H, d); 7.04 (4H, d); 4.25 (2H, t); 3.71 (2H, m); 3.65 (4H, m); 3.55(2H, t); 3.36 (2H, t); 3.31 (12H, s); 3.30 (2H, m); 3.07 (2H, m); 2.49(4H; m); 2.44 (2H, t); 2.15 (2H, q).

[4-(Boc-Aminoethyl-PEG11)-6,9,15-triaza-1,12-dioxa-5,10,14-trioxopentaundecanyl)Leuko-MG-NHBOC19

O-[2-Boc-amino)-ethyl]-O′[2-(diglycolyl-amino)ethyl]decaethylene glycolP11 (152 mg, 0.2 mmol) were dissolved in dry DMF (0.5 mL). TSTU (66 mg,2.2 mmol) and DEA (39 μL, 2.2 mmol) were added. The reaction mixture waskept overnight at room temperature.[4-(9-Amino6-aza-1-oxa-5-oxo-nonyl)phenyl]bis[4-(dimethylamino)phenyl-methane(100 mg, 0.2 mmol) 16 was dissolved in dry DMF (0.2 mL) and added to theactive ester of P11. The reaction mixture was kept at room temperaturefor 3 hrs. The solvent was removed under vacuum and the residue purifiedon silicagel (eluent: ethyl acetate/10-30% methanol). MW: C₆₂H₉₁N₆O₁₈1208.5 g/mol. Yield: 150 mg (62%). (MG(H)—O—(CH₂)3-CONH—C₂H₄—NHCO—CH₂—O—CH₂—CONH—(C₂H₄O)₁₁—C₂H₂—NH₂NHBOC; ¹H-NMR (CD3CN):7.28 (1H, d), 7.17 (2H, d), 7.11 (1H, d), 7.02 (2H, d), 6.80 (2H, dd),6.67 (2H, dd), 5.4 (1H, s), 3.95 (2H, m), 3.91 (2H, d), 3.88 (2H, s),3.54 (42H, m), 3.45 (2H, t), 3.39 (2H, m), 3.29 (2H, m), 3.27 (2H, m),2.89 (12H, s), 2.30 (2H, m), 2.00 (2H, m), 1.40 (9H, s).

MG-11P 20

Leuko-MG-11P-NHBoc 19 (150 mg, 0.124 mmol) was dissolved in 25 mLacetonitrile. The reaction mixture was heated to 50 C.Tetrachloroquinone (100 mg, 0.4 mmol) was dissolved in 30 mL boilingacetonitrile and dropwise added under stirring to the leukobase. Thedark green dye formation was followed by ¹H-NMR control. After severalhours the reaction mixture was cooled to room temperature. Theacetonitrile was removed and the reaction mixture was partitionedbetween water (150 mL) and ethyl acetate (50 mL). The water phase wasseparated and washed several times with ethyl acetate. The water phasewas concentrated to give a green residue. Hydrochloric acid in ethanol(2 mL of a 20% solution) was added and the reaction mixture was stirredfor 1 hr. The reaction mixture was concentrated to dryness to give 60 mgof a green powder. Yield: 53%MG-O—(CH₂)₃—CONH—C₂H₄—NHCO—CH₂—O—CH₂—CONH—(C₂H₄O)₁₁—C₂H₂—NH₂ ¹H-NMR(MeOD): 7.43 (4H, d); 7.36 (2H, d), 7.17 (2H, d), 7.05 (4H, d), 4.20(2H, t), 4.06 (2H, d); 4.05 (2H, d), 3.77 (2H, t), 3.78-3.58 (44H, m),3.45 (2H, t), 3.32 (12H, s), 3.18 (2H, t), 2.45 (2H, t), 2.15 (2H, m).C₅₇H₉₁N₆O₁₆+MS/M+=1116.53

MG-11P-Biotin

MG-11p 20 (11 mg; 0.01 mmol) was dissolved in 0.2 mL DMSO. A solution ofBiotinNHS ester (6.5 mg; 0.02 mmol) in 0.1 mL DMSO was added followed by0.02 ml, of DEA (1 mMol in DMSO). The reaction mixture was stirred for 2hrs at room temperature, then passed through a short column of neutralaluminum oxide. DMSO and DEA were eluted with chloroform. The productwas eluted with chloroform/10-20% methanol to give 8 mg of a greensolid. Yield: 58%.

MG-O—(CH₂)₃—CONH—C₂H₄—NHCO—CH₂—O—CH₂—CONH—(C₂H₄O)₁₁—C₂H₄—NH-Biotin¹H-NMR (MeOD): 7.43 (4H, d), 7.37 (2H, d), 7.18 (2H, d), 7.06 (4H, d),4.51 (1H, m), 4.32 (1H, m), 4.21 (2H, m), 4.06 (2H, s), 4.05 (2H, s),3.65 (44H, m), 3.55 (2H, m), 3.47 (2H, m), 3.38 (6H, m), 3.32 (12H, s),3.25 (4H, m), 3.06 (1H, m), 2.93 (3H, m), 2.45 (2H, m), 2.22 (2H, m),2.16 (2H, qui). C₆₇H₁₀₅C1 N₈O₁₈S+; MS/M+=1342.60

Biotin-PEG5000-MG

MG-2P 18 (5 mg; 0.01 mmol) and Biotin-PEG5000-NHS ester 8 (NektarTherapeutics) (50 mg; 0.01 mmol) were dissolved in 0.2 mL of DMF and0.01 mL of DEA (1 mMol in DMF) was added. The reaction mixture wasstirred for 1 hr. The reaction mixture was passed through a short columnof neutral aluminum oxide. DMF and DEA were eluted with chloroform. Theproduct was eluted with chloroform/10-20% methanol. Mn=5747.8.

N-[4-[(4-Carboxyphenyl)[4-(dimethylamino)phenyl]methylene]-2,5-cyclohexadienlylidene]-N-methyl-methanaminiumchloride 21

All publications and patents mentioned herein, included those listedbelow, are hereby incorporated by reference in their entirety as if eachindividual publication or patent was specifically and individuallyincorporated by reference. In case of conflict, the present application,including any definitions herein, will control.

Also incorporated by reference are the following: U.S. Pat. Nos.5,334,537; 5,998,142; 6,287,765; 6,297,059; 6,331,394; 6,358,710; WO02/23188; WO 02/18952; Bark and Hahn, Methods 20:429-435 (2000); Barkeret al., Anal. Chem. 71: 1767-1172 (1999); Barker et al., Anal. Chem. 71:2071-2075 (1999); Bradbury, Nature Biotechnology 19: 528-529 (2001);Benhar, Biotechnology Advances 19: 1-33 (2001); Carrero and Voss, J.Biol. Chem. 271: 53325337 (1996); Chamberlain and Hahn, Traffic 1:755-762 (2000); Chen et al., Nature Biotechnology 19: 537-542 (2001);Hahn et al., J. Biol. Chem. 265: 20335-20345 (1990); Marks et al., J.Mol. Biol. 222: 581-597 (1991); Post et al., J. Biol. Chem. 269:12880-12887 (1994); Post et al., Mol. Biol. Cell 6: 1755-1768 (1995);Ramjiawan et al., Cancer 89: 1134-44 (2000); Skerra, J. Mol.Recognition. 13: 167-187 (2000); and Sumner et al., Analyst 127: 11-16(2002)

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Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. While specificembodiments of the subject invention have been discussed, the abovespecification is illustrative and not restrictive. Many variations ofthe invention will become apparent to those skilled in the art uponreview of this specification. The full scope of the invention should bedetermined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations. Suchequivalents are intended to be encompassed by the following claims.

We claim:
 1. A ligand-dye complex, comprising a cognate ligand of a dyenon covalently bound to the dye, wherein the cognate ligand comprises anscFv molecule consisting of a polypeptide sequence having at least 90%sequence identity to the polypeptide of SEQ ID NO: 3, wherein the dyecomprises one of a thiazole orange or a thiazole orange analog thatbinds the scFv molecule, and wherein the bound dye and ligand exhibit anincrease in detectable fluorescence signal at least ten times greaterthan the detectable fluorescence signal of the dye when not bound to theligand.
 2. The ligand-dye complex of claim 1, wherein the ligand-dyecomplex further comprises the scFv molecule bound to a protein.
 3. Theligand-dye complex of claim 1, wherein the bound dye and ligand exhibitan extinction coefficient of greater than 30,000 and a maximumabsorption at a wave length of greater than 350 nm.
 4. The ligand-dyecomplex of claim 1, wherein the bound dye emits photons having a changedpolarization upon binding the ligand.
 5. The ligand-dye complex of claim4 wherein the polarization change is greater than or equal to 20%. 6.The ligand-dye complex of claim 1, wherein the dye of the bound dye andligand exhibits an increased emission wave length of at least 10 nm. 7.The ligand-dye complex of claim 1, wherein the ligand is fixed to asubstrate.
 8. The ligand-dye complex of claim 7, wherein the substratecomprises molecules of formula X(a)-R(b)-Y(c), wherein R is a spacer, Xis a functional group that binds R to a surface, Y is a functional groupfor binding to the ligand, (a) is an integer from 0 to about 4, (b) isan integer from 0 or 1, and (c) is an integer not equal to
 0. 9. Theligand-dye complex of claim 1, wherein the dye is thiazole orange analogTO1 comprising the following general structure:

wherein R is selected from the group consisting of a fluorescent labeloptionally comprising a linker, a photoreactive group, and a reactivegroup.
 10. The ligand-dye complex of claim 1, wherein the dye isthiazole orange analog TO1 comprising the following general structure:

wherein R is a group that comprises biotin, a hapten, or a His-tag,optionally with a linker.
 11. The ligand-dye complex of claim 9, whereinthe thiazole orange analog TO1 has the following structure:


12. The ligand-dye complex of claim 9, wherein the ligand-dye complexoccupies a microenvironment and wherein the dye provides the increase indetectable fluorescence signal in response to pH, polarity, restriction,or mobility properties within the microenvironment.
 13. The ligand-dyecomplex of claim 1, wherein the dye is thiazole orange analog TO1comprising the following general structure:

wherein R is a moiety that controls water solubility and non-specificbinding.
 14. The ligand-dye complex of claim 1, wherein the dye isthiazole orange analog TO1 comprising the following general structure:

wherein R is a moiety that controls the ability of the dye to enter acellular compartment.
 15. The ligand-dye complex of claim 1, wherein thedye is thiazole orange analog TO1 comprising the following generalstructure:

wherein R is a group that comprises a moiety to facilitate isolation ofthe ligand.
 16. The ligand-dye complex of claim 1, wherein the scFvmolecule has the polypeptide sequence of SEQ ID NO:
 7. 17. Theligand-dye complex of claim 1, wherein the scFv molecule has thepolypeptide sequence of SEQ ID NO:
 9. 18. A ligand-dye complexcomprising: an scFv molecule having complementarity determining regionsof SEQ ID NOs 76, 77 and 78 noncovalently bound to a dye comprising oneof a thiazole orange or a thiazole orange analog that binds the scFvmolecule; wherein the bound dye and ligand exhibit an increase indetectable fluorescence signal at least ten times greater than thedetectable fluorescence signal of the dye when not bound to the ligand.19. The ligand-dye complex of claim 18 wherein the dye is thiazoleorange analog TO1 comprising the following general structure:

wherein R is selected from the group consisting of a fluorescent labeloptionally comprising a linker, a photoreactive group, and a reactivegroup.
 20. The ligand-dye complex of claim 18 wherein the dye has thefollowing structure:


21. A method of forming a ligand-dye complex for use in detectingexpression of a gene in a cell, the method comprising: adding a vectorto a cell, wherein the vector comprises a nucleic acid gene encoding ascFv molecule consisting of a polypeptide sequence having at least 90%sequence identity to the polypeptide of SEQ ID NO: 3; causing expressionof the gene to produce a gene product comprising the scFv molecule;adding dye to the cell to bind to the gene product, wherein the dyecomprises one of thiazole orange or a thiazole orange analog comprisingthe following general structure:

wherein R is selected from the group consisting of a moiety thatcontrols water solubility and non-specific binding, a moiety thatcontrols the ability of the dye to enter a cellular compartment, a groupthat comprises, optionally with a linker, biotin, a hapten, a His-tag,or a moiety to facilitate isolation of the ligand, a fluorescent labeloptionally comprising a linker, a photoreactive group, and a reactivegroup.
 22. The method of claim 21, wherein the gene further encodes asecond protein, and wherein the gene product comprises a fusion proteincomprising the scFv molecule and the second protein.
 23. The method ofclaim 22, wherein the dye is substantially excluded from an interior ofthe cell, and wherein fluorescence is detected only when a portion ofthe second protein that is fused to the scFv molecule is exposed at asurface of the cell.
 24. The method of claim 21, wherein the dye isthiazole orange analog TO1-R, and wherein R is a moiety that controlsthe ability of the dye to enter a cellular compartment.
 25. A method offorming and using a ligand-dye complex for detecting interaction of twoproteins in a cell comprising: adding a vector to a cell, wherein thevector comprises a nucleic acid gene encoding a first protein fused to afirst portion of a scFv polypeptide sequence having at least 90%sequence identity to the polypeptide of SEQ ID NO: 3; adding a vector toa cell, wherein the vector comprises a nucleic acid gene encoding asecond protein fused to a second portion of the scFv polypeptidesequence; causing expression of the genes to produce fusion proteinscomprised of the first and second proteins fused to first and secondportions, respectively, of the scFv polypeptide; and adding dye to thecell, wherein the dye comprises one of thiazole orange or a thiazoleorange analog comprising the following general structure:

wherein R is selected from the group consisting of a moiety thatcontrols water solubility and non-specific binding, a moiety thatcontrols the ability of the dye to enter a cellular compartment, a groupthat comprises, optionally with a linker, biotin, a hapten, a His-tag,or a moiety to facilitate isolation of the ligand, a fluorescent labeloptionally comprising a linker, a photoreactive group, and a reactivegroup; and detecting a fluorescence signal indicative of the dye bindingto the fusion proteins, wherein the fluorescence signal is at least tentimes greater than the detectable fluorescence signal of the dye whennot bound to the fusion proteins.
 26. The method of claim 25, whereinthe first scFv polypeptide portion and the second scFv polypeptideportion interact to form a scFv molecule consisting of a polypeptidesequence having at least 90%-sequence identity to the polypeptide of SEQID NO 3, and wherein fluorescence is detectable when interaction betweenthe first protein and the second protein brings the first scFvpolypeptide portion into proximity of the second scFv polypeptideportion.